Methods and compositions for controlling insects

ABSTRACT

Compositions and methods for controlling insects by co-expressing an amino acid oxidase and a second enzyme that provides insecticidal activity when present in a mixture with the amino acid oxidase are disclosed. Also disclosed are DNA and protein sequences, and transformed microorganisms and plants useful for achieving such insect control.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. ProvisionalApplication Serial No. 60/044,504, filed Apr. 21, 1997.

FIELD OF THE INVENTION

[0002] This invention relates to compositions and methods forcontrolling coleopteran insects by use of two proteins in combinationwhich may be applied directly to the plant or produced thereon bymicroorganisms or by genetically modifying the plant to produce theproteins, to genes encoding these proteins, to methods for identifyingsuch genes and proteins, and to recombinant microorganisms and plantscapable of expressing these genes for use in controlling plantinfestation by the target coleopteran insects.

BACKGROUND OF THE INVENTION

[0003] The control of insect pests by naturally occurring proteins is awell established practice. The most commonly used insect controlproteins are the endotoxins derived from Bacillus thuringiensis (B.t.)that are used to control both lepidopteran and coleopteran insect pests.Expression of these proteins in transgenic plants also confersprotection against certain insect pests (Barton et al., 1987; Fischhoffet al., 1987 Perlak et al., 1990; Vaeck et al., 1987).

[0004] A variety of insect pests that cause significant economic losseswere not previously known to be controlled by B.t. endotoxins. Bollweevil (BWV), Anthonomus grandis, corn rootworm (CRW), Diabroticla spp.,and wireworm (WW), Melanotus spp. are examples of coleopteran insectpests that inflict significant crop damage yet, until recently were notknown to be controlled by known B.t. endotoxins. Thus, it would beuseful to identify new insecticidal proteins which, alone or incombination, are able to control these coleopteran insects. Furthermore,it would be useful to identify new insecticidal proteins with differentmodes of action to delay the development of B.t. endotoxin resistance incoleopteran pests such as the Colorado potato beetle (CPB), Leptinotarsadecemlineata (Say), that are currently controlled by certain B.t.endotoxins (Krieg et al., 1983).

[0005] Preparations of enzymes from several different sources areavailable from Sigma Chemical Company (St. Louis, Mo.) and othersuppliers. Amino acid oxidases can also be obtained from sourcesincluding, but not limited to snake venom, mammalian, and avian sources(Bright and Porter, 1975). Lysine and other amino acid oxidases (E.C.1.4.3.2) are naturally produced by micro-organisms such as Trichodermasp., Neurospora sp., Penicillium sp., and Proteus sp. (Kusakabe et al.,1979; 1980; Niederman and Lerch, 1990; Knight, 1948; Stumpf and Green,1944). Although lysine oxidase has been shown to have antitumor activity(Kusakabe et al., 1979; Id., 1980), there have been no reports ofinsecticidal activity associated with this enzyme. Also, there have beenno reports of insecticidal activity being associated with an amino acidoxidase enzyme when combined with any other compound. However, we haveunexpectedly found that a composition comprising a lysine oxidase and apreviously unidentified M_(r) 50,000 protein yield potent insecticidalactivity when combined in a mixture and ingested by an insect. The M_(r)50,000 protein is described herein as a tedanalactam synthase shownherein to have at is least one enzyme activity in whichΔ1-piperideine-2-carboxylate is converted to tedanalactam. Describedherein are methods for using a combination of lysine oxidase andtedanalactam synthase to control infestation of plants by insect pests.

SUMMARY OF THE INVENTION

[0006] This invention relates generally to novel compositions andmethods for the control of undesired insects. It is therefore aparticular object of the present invention to present materials andmethods used in the preparation of compositions and plants capable ofcontrolling insect infestation when ingested by the insect. It is alsoan object of the present invention to provide protein compositionscapable of controlling BWV, CRW, WW, CPB or other insect pests, andgenes useful in producing such proteins. It is a further object of thepresent invention to provide genetic constructs for and methods ofinserting such genetic material into microorganisms and plant cells. Itis another object of the present invention to provide transformedmicroorganisms and plants containing such genetic material. Stillanother object of this invention relates to methods and reagents such aspolynucleotides and antibodies, and the use of such methods and reagentsin kit form, for detecting the individual molecules which comprise theactive compositions as noted herein. In addition, variants of themolecules which comprise the active compositions are also contemplatedby this invention.

[0007] Among the several advantages found to be achieved by the presentinvention, therefore, may be noted the provision of a compositioncontaining at least two proteins, lysine oxidase enzyme and tedanalactamsynthase enzyme, which is capable of controlling insects, particularlycoleopteran insects. These two proteins cause mortality and stunting oflarvae of coleopteran insects when co-ingested. The proteins may beapplied directly to plants or introduced in other ways such as throughthe application of plant-colonizing microorganisms or by transformedplants generated using recombinant DNA methods wherein the recombinantplants express genes encoding these enzymes.

[0008] In accomplishing the foregoing, there is provided, in accordancewith one aspect of the present invention, a method of controlling insectinfestation of plants comprising providing a composition containing atleast a lysine or amino acid oxidase along with a second enzyme, whichis preferably a tedanalactam synthase, for ingestion by the insect. Itis apparent that neither protein alone is able to confer anyinsecticidal activity. However, it is the combination of the amino acidoxidase along with the second enzyme which is effective in conferinginsecticidal activity upon ingestion of such a composition by an insect.The composition, upon ingestion by the insect, contains a sufficientinsecticidal amount of the proteins, such that the insect is unable tosurvive or is rendered incapable of causing further damage to a plant towhich the composition has been applied. The composition contains a firstenzyme which is a lysine oxidase enzyme, and a second enzyme which iscapable of converting Δ1-piperideine-2-carboxylate to tedanalactam. Theproteins in the composition are preferably isolated from extracts offungal species fermentations in which the extracts have been shown toexhibit insecticidal activity. The fungal species herein which producesan insecticidally effective extract composition was determined to be aTrichoderma species of fungi, and in particular a Trichoderma viride.Another Monsanto Company fungal isolate was designated as Trichodermasp. F22844 also produces an insecticidally effective extractcomposition. The genes encoding the proteins in the illustrativecomposition are therefore preferably isolated from a Trichoderma speciesof fungi, however, other uncharacterized fungal species are believed tocontain at least a lysine oxidase gene and a second protein which, incombination provide efficacious insecticidal activity.

[0009] The composition can contain as the second enzyme a protein whichis approximately 50,000 Da, which is also recognized by one skilled inthe art as a protein or enzyme which is approximately M_(r) 50,000. Itis believed that the second enzyme can be isolated from any number ofspecies, however it is preferably isolated from a species which producescompounds which exhibit coleopteran insecticidal activity, and morepreferably isolated from a fungal species. It is also believed that anyfungal species which exhibits coleopteran insecticidal activity and alsoproduces a lysine oxidase may also produce a second enzyme which incombination with the lysine oxidase confers effective insecticidalactivity when ingested by target insect. Furthermore, any species whichproduces a lysine oxidase and which also contains a gene whichhybridizes under stringent conditions to a Trichoderma species geneencoding an approximately M_(r) 50,000 Da protein which convertsΔ1-piperideine-2-carboxylate to tedanalactam may confer effectiveinsecticidal activity when a composition containing both enzymes isingested by a target insect. The property of convertingΔ1-piperideine-2-carboxylate to tedanalactam may be independent of theproperty which, in combination with an amino acid oxidase confersinsecticidal activity upon the composition when ingested by the insect.It is intended that the composition not be limited to a combination ofan amino acid oxidase and a tedanalactam synthase, but conceivably couldalso include the combination of a gene encoding an amino acid oxidaseand a gene encoding a tedanalactam synthase, together with all necessarygenetic regulatory elements required for expression, includingrepression and activation, transcription and translation, andpost-transcriptional and post-translational modification signalsincorporated therein. The genes as described could also be presenteither alone or in combination with each other on a single replicon.

[0010] The composition which confers coleopteran insecticidal activityis directed preferably to coleopteran species selected from the groupconsisting of Diabrotica species, Melanotus species, Leptinotarsaspecies, and Anthonomus species. Moreover, the composition is directedto controlling insects selected from the group consisting of boll weevil(BWV), corn rootworm (CRW), corn wireworm (WW), and the Colorado potatobeetle (CPB).

[0011] The compositions in particular can contain the indicated enzymesin a mixture in which the molar ratios of the two enzymes are generallysuch that effective insect control is manifested. Insect control can beeffected when the amino acid oxidase and the tedanalactam synthase arepresent within the composition in molar ratios of about 100:1 to about1:1 respectively, or when the ratios are about 10:1 to about 1:1,respectively, or when the ratios of the two proteins are present fromabout 1:10 to about 1: 1, or when the ratios of the two proteins arepresent from about 1:100 to about 1:1, respectively. In addition,effective concentrations of these proteins in a composition in which theproteins are each present from about one part per million to about 10parts per million are effective in conferring insecticidal activity andcontrol. The most effective insecticidal activity is conferred when theproteins are each present in a composition from about one part permillion to about 20 parts per million.

[0012] Another aspect of the present invention provides the structuralgenes which encode the enzymes which are the active components in theinsecticidal compositions. Briefly, the genes can be isolated fromgenomic DNA and from cDNA molecules which are obtained by isolating mRNAfrom species which are shown to produce these enzymes. The structuralgenes encoding these enzymes, which may also be isolated as proenzymesor precursor proteins, preferably are identified by first isolating theactive components or enzymes from extracts of organisms which producethese enzymes. Isolated enzymes can be digested with proteolyticenzymes, and amino acid sequences of proteolytic peptide fragments canbe characterized. Redundant nucleotide probes corresponding to thecharacterized peptide fragments can be produced based on the deducedamino acid sequences, and used as probes or primers for identifying oramplifying particular segments of mRNA, cDNA, or genomicpolynucleotides. Full length mRNA, full length cDNA, and uninterruptedfull length genes can be further identified and isolated.

[0013] In accordance with other aspects of the present invention, thereare provided methods and compositions for producing geneticallytransformed plants which express an amount of a lysine or other aminoacid oxidase along with a second enzyme or tedanalactam synthaseeffective to control coleopteran insects. Recombinant plasmids have beenproduced which contain regulatory elements which function in plants forproducing messenger RNA molecules, from which the proteins of thepresent invention are translated. Expression cassettes are disclosedwhich contain various elements alone or in combination for enabling theproduction of the amino acid oxidase or the tedanalactam synthase.Specifically, the amino acid oxidase gene is provided in a cassettecomprising a polynucleotide sequence flanked 5′ by a promoter whichfunctions in plants to cause the production of an RNA sequence isoperably linked to an intron and a DNA sequence which functions inplants as a targeting signal or transit peptide and flanked 3′ by a DNAsequence which functions in plants to cause the addition of a 3′non-translated polyadenylated nucleotide sequence to the 3′ end of theRNA is fused 3′ to the amino acid oxidase gene so that the expression ofthe cassette is under the control of the promoter. There are numerousalternatives to this construction, some of which are provided herein inspecific examples. For example, the intron and targeting sequence can bereplaced by a 5′ non-translated leader sequence; or the non-translatedleader can be removed; the intron can be inserted between thenon-translated leader and the oxidase gene or between the leader and thetargeting sequence. The tedanalactam synthase can be assembled in asimilar fashion, and specific examples are provided herein. Anexpression cassette for producing an amino acid oxidase can be combinedinto a single vector along with a cassette for producing a tedanalactamsynthase so that delivery of both cassettes for simultaneous expressioneither in a plant or other organism such as a bacterium or fungi is alsocontemplated. Also, in a plant it is possible to express one of thecassettes in one tissue type, for example in roots, and express theother cassette in another tissue type, for example in leaves. It mayalso be possible to produce the proteins separately temporally orspatially, but in the same tissue type. For example, expression of onecassette in young leaves and the other cassette later in the same leavesis contemplated, however co-expression is normally desirable. Theexpression cassette can be designed to function in plants by using plantspecific regulatory elements such as promoters, introns, targetingsequences, non-translated leaders, and 3′ polyadenylation sequences. Theexpression cassette can also be designed to function in prokaryoticsystems as contemplated and described herein, also by using prokaryoticspecific regulatory elements. The cassettes described herein can beinserted into plants by high velocity DNA coated particle projectilebombardment, by naked DNA protoplast transformation, or by bacterialmediated methods known in the art.

[0014] In describing this particular embodiment of the invention, itshould be understood that expression of the amino acid oxidase, whichcan also be a precursor or proenzyme, and tedanalactam synthase can becontrolled by two independent promoters from two separate andindependent transcriptional units. It should also be understood that asingle promoter could be used to drive expression of a singletranscription unit containing an in frame translational fusion of bothproteins. The hybrid polyprotein could then be post-translationallycleaved to yield both proteins by previously described schemes (Halpinand Ryan, WO 95/17514). Another advantage achieved by the presentinvention provides a peptide fusion to be produced from the genesencoding the two enzymes wherein the coding sequences of the two genesare fused in frame to allow for the expression of a recombinant geneencoding an in-frame translational peptide fusion of the amino acidoxidase and the tedanalactam synthase. The fusion can be one in whicheither enzyme is amino terminal with respect to the other. The fusioncan be post-translationally cleaved by a plant endogenous endoproteaseto produce an insecticidally active composition in the plant tissues sothat lysine oxidase and tedanalactam synthase are present as separateand individual molecules. Alternatively, the fusion can bepost-translationally cleaved by an endogenous insect endoprotease,generally found within the midgut of contemplated insect targets, sothat the cleavage of the fusion protein produces an insecticidallyactive composition while within the midgut of the feeding insect.

[0015] In keeping with this aspect of the present invention, is theprovision for a variety of promoters for transcriptional initiation andexpression of the contemplated genes, in particular in plants. A numberof promoters which are active in plant cells have been described in theliterature. Such promoters may be obtained from plants or plant virusesand include, but are not limited to the nopaline synthase (NOS) andoctopine synthase (OCS) promoters, which are carried on tumor-inducingplasmids generally found within virulent and non-virulent strains ofAgrobacterium tumefaciens, the cauliflower mosaic virus (CaMV) 19S and35S promoters, the light-inducible ribulose 1,5-bisphosphate carboxylasesmall subunit promoter(ssRUBISCO), and the Figwort Mosaic Virus 35Spromoter (FMV). All of these promoters have been used to create varioustypes of DNA constructs which have been expressed in plants (see forexample Barry et al. U.S. Pat. No. 5,463,175, which is hereinincorporated by reference). Particularly desirable promoters which arecontemplated because of their constitutive nature are the CauliflowerMosaic Virus 35S (CaMV35S) and the Figwort Mosaic Virus 35S (FMV35S)promoters which have previously been shown to produce high levels ofexpression in most plant organs. Other preferred promoters are rootenhanced or root tissue specific promoters such as the CauliflowerMosaic Virus derived AS4 promoter (also designated as the 4×as-1 or the4as-1 promoter), the tobacco RB7 promoter, or the rice RC2 promoter (Lamet al., 1991; Yamamoto et al., 1991; Xu et al., 1995). The root enhancedor root tissue specific promoters would be particularly preferred forthe control of corn rootworm (Diabrotica spp.) in transgenic cornplants. Other promoters are also contemplated which would direct tissuespecific targeted expression are also contemplated, for example intissue such as leaves, meristem, flower, fruit and organs ofreproductive character. IN addition, chimeric promoters are alsoenvisioned.

[0016] Other expression regulatory elements are considered to be ofimportance, especially in contemplation of transformed plant tissue ortransformed plant cell expression. These elements comprise at leastnon-translated sequences and introns. For example, transcriptionalevents leading to RNA production from the contemplated DNA constructs asset forth herein could contain 5′ non-translated leader sequences. Thesesequences can be derived from new or existing promoters which areselected for gene expression, and can be specifically modified so as toincrease translational efficiency of the mRNA. A plant gene leadersequence which has been shown to be particularly useful in the presentinvention is the petunia heat shock protein 70 leader (hsp70)(Winter etal., 1988). It has also been shown that introns are preferred foroptimum expression in monocotyledenous plants. Any number of intronscould function for this purpose, and without intending any limitationthese could be selected from the group consisting of the maize hsp70intron and the rice actin intron. Another nontranslated regulatoryelement of particular importance in plant systems are DNA sequenceswhich function in plants to cause the addition of a 3′ non-translatedpolyadenylated nucleotide sequence to the 3′ end of an RNA sequenceproduced as a result of transcription from an indicated promotersequence. These particular non-translated sequences are also commonlyknown as polyadenylation sequences or signals.

[0017] A further embodiment provides for the targeted delivery of a geneproduct to a particular organellar compartment, such as a vacuole, amitochondrion, a chloroplast, a plastid, an endoplasmic reticulumcompartment, a Golgi compartment, or even the nuclear or nucleolardomains. Particular peptide sequences have been shown to be necessary inobtaining efficient delivery of protein products to these sites, inparticular signal peptides, signal sequences, and targeting sequences.Targeting signals or transit peptides contemplated herein, withoutintending to be limited to these, can be selected from the groupconsisting of a rice malate dehydrogenase amino terminal peroxisomaltargeting signal and a maize ATP synthase beta subunit mitochondrialtransit peptide.

[0018] A further embodiment of the present invention provides for theinsertion of one or more of the contemplated expression cassettes alongwith any expression regulatory elements into the genome of a plant cellto form a stable recombinant plant cell. In one embodiment the DNAinserted into the plant genome would be comprised of a single cassetteencoding a fusion peptide formed from the gene fusions described above,along with regulatory elements necessary for expression of the genefusion. In another embodiment, the DNA inserted into the genome would becomprised of a single cassette encoding only an amino acid oxidase or atedanalactam synthase, along with any regulatory elements necessary forexpression of the gene. In the preferred embodiment, the DNA insertedinto the plant genome would be comprised of a first gene cassetteencoding an amino acid oxidase in which expression was controlled by afirst promoter, and a second gene cassette encoding a tedanalactamsynthase in which expression was controlled by a second promoter, alongwith any other necessary regulatory elements for expression of eithergene. This embodiment contemplates that the first and the secondpromoters can be identical in sequence and function, or they can bedifferent from each other, so long as each gene is expressed in anamount which provides a composition for controlling insect infestationof plants comprising a mixture of the enzymes when the mixture isingested by a susceptible insect.

[0019] A further embodiment of the present invention provides methodsfor generating plants which express an insecticidally effective amountof a lysine or amino acid oxidase or proenzyme along with a tedanalactamsynthase. The methods utilize contemplated DAN expression cassettesdesigned for producing either or both enzymes separately or incombination inserted into plasmids. The plasmid DNAs can be directlyinserted into the genome of a plant by mechanical approaches, such asbiolistic methods or by protoplast fusion techniques. A preferred methodutilizes Agrobacterium mediated double border plant transformation,preferably using a DNA vector containing the desired expression cassetteor cassettes flanked by Ti plasmid border recombination sequences inorder to introduce the desired genes into the plant genome. Thetransformation procedure generally produces events which providestransformed plant cells selected on solid or in liquid media using anynumber of selectable markers known in the art, preferably glyphosateselectable markers such as GOX or EPSPS, antibiotic selectable markers,or others. Transformed cells obtained using these methods can be furtherregenerated to produce stably transformed genetically engineered plantswhich express insecticidally effective amounts of the amino acid oxidaseor the tedanalactam synthase.

[0020] There is also provided, in accordance with another aspect of thepresent invention, transformed bacterial and plant cells that containDNA comprised of the expressible gene cassettes as described above,along with appropriate control sequences necessary to provide fordesired and appropriate expression of the coding sequences, producinginsecticidally effective amounts of the enzymes. The control sequencescan be any known in the art to function in a particular cell ororganelle type. It is contemplated that the genes herein can beexpressed in bacterial systems, plant nucleolar compartments, plantnuclear compartments, and in plant mitochondrial and chloroplastcompartments. While particular examples of using the invention describedherein to control corn rootworm in corn or Colorado potato beetle inpotato are provided, it is understood that the methods of this inventioncould be applied to provide insect protection, and more preferablycoleopteran insect protection, to plant species from the generaFabaceae, Medicago, Trifolium, Vigna, Daucus, Arabidopsis, Brassica,Raphanus, Sinapis, Lycopersicon, Capsicum, Solanum, Nicotiana,Helianthus, Bromus, Asparagus, Panicum, Pennisetum, Cucumis, Lolium,Glycine, Triticum, Gossypium and Zea. In addition, forestry crop speciesfrom the genera Pinus, Populus, Eucalyptus, Acacia, Silex, and Larix arealso prone to important coleopteran pest infestation which may becontrolled by the methods and compositions described herein. Also, turfgrass species such as St. Augustine (Stenotaphrum secundatum), Kentuckyblue grass (Poa pratensis), and creeping bentgrass (Agrostisstolonifera) among others are susceptible to coleopteran pests such aswhite grub and the like which may also be controlled by the presentinvention. Insect pests which infest Roses (Rosa) and perennials such asBegonia, Pelagonium, Imaptiens, Tagetes, Viola, Petunia and Catharanthusand the like may also be subjects of the present invention.

FIGURE LEGENDS

[0021]FIG. 1 represents a plasmid map of pMON25061 which is a planttransformation vector containing a neomycin phosphotransferaseselectable marker under the control of a cauliflower mosaic virus 35Spromoter; and a tedanalactam synthase gene and lysine oxidase gene eachunder the control of separate root enhanced 4AS1 promoters.

[0022]FIG. 2 represents a plasmid map of pMON25049 which is anAgrobacterium mediated double border plant transformation vectorcontaining a neomycin phosphotransferase selectable marker under thecontrol of a cauliflower mosaic virus 35S promoter; a lysine oxidasegene fused to a petunia hsp70 leader sequence under the control of afigwort mosaic virus promoter; and a tedanalactam synthase gene fused toa petunia hsp70 leader sequence under the control of a figwort mosaicvirus promoter.

[0023]FIG. 3 represents a plasmid map of pMON23671 which contains an 800base pair cDNA fragment representing a portion of a lysine oxidase gene.

[0024]FIG. 4 represents a plasmid map of pMON23683 which contains a 650base pair cDNA fragment representing a portion of the 3′ end of a lysineoxidase gene.

[0025]FIG. 5 represents a plasmid map of pMON23681which contains a 300base pair cDNA fragment representing a portion of the 5′ end of a lysineoxidase gene.

[0026]FIG. 6 represents a plasmid map of pMON23680 which contains agenomic DNA fragment encoding a lysine oxidase gene.

[0027]FIG. 7 represents a plasmid map of pMON23684 which contains agenomic DNA fragment encoding a lysine oxidase gene.

[0028]FIG. 8 represents a plasmid map of pMON25421 which contains a cDNAfragment representing a partial coding sequence of a tedanalactamsynthase gene.

[0029]FIG. 9 represents a plasmid map of pMON25422 which contains a cDNAfragment representing a partial coding sequence of a tedanalactamsynthase gene.

[0030]FIG. 10 represents a plasmid map of pMON25424 which contains afull length cDNA representing the coding sequence of a tedanalactamsynthase gene.

[0031]FIG. 11 represents a plasmid map of pMON25030 which represents ayeast expression vector containing a DNA fragment encoding a lysineoxidase.

[0032]FIG. 12 represents a plasmid map of pMON25428 which contains acDNA fragment encoding a tedanalactam synthase under the control of anarabinose inducible promoter.

[0033]FIG. 13 represents a plasmid map of pMON19469 which is a monocotexpression vector containing a cauliflower mosaic virus 35S promoter, anhsp70 intron, and a nopaline synthase polyadenylation sequence.

[0034]FIG. 14 represents a plasmid map of pMON25040 which contains a DNAsequence encoding a lysine oxidase variant under the control of acauliflower mosaic virus 35S promoter.

[0035]FIG. 15 represents a plasmid map of pMON15786 which is a planttransient expression vector containing a neomycin phosphotransferasecoding sequence fused to an hsp70 intron, under the control of acauliflower mosaic virus 35S promoter.

[0036]FIG. 16 represents a plasmid map of pMON25041 which contains a DNAfragment encoding a lysine oxidase variant under the control of acauliflower mosaic virus 35S promoter.

[0037]FIG. 17 represents a plasmid map of pMON30411 which is a planttransformation vector containing a tedanalactam synthase variant geneunder the control of a cauliflower mosaic virus 35S promoter.

[0038]FIG. 18 represents a plasmid map of pMON30410 which is a planttransformation vector containing a tedanalactam synthase gene under thecontrol of a cauliflower mosaic virus 35S promoter.

[0039]FIG. 19 represents a plasmid map of pMON30417 which is a planttransformation vector containing a neomycin phosphotransferaseselectable marker, a tedanalactam synthase gene, and a lysine oxidasegene each under the control of a separate cauliflower mosaic virus 35Spromoter.

[0040]FIG. 20 represents a plasmid map of pMON25058 which is a planttransient expression vector containing a lysine oxidase gene fused to anhsp70 intron under the control of a root tissue enhanced promoter.

[0041]FIG. 21 represents a plasmid map of pMON25043 which is a planttransient expression vector containing a tedanalactam synthase genefused to an hsp70 leader under the control of a figwort mosaic viruspromoter.

[0042]FIG. 22 represents a plasmid map of pMON10098 which is anAgrobacterium mediated double border plant transformation vectorcontaining a neomycin phosphotransferase selectable marker under thecontrol of a cauliflower mosaic virus 35S promoter.

[0043]FIG. 23 represents a plasmid map of pMON25046 which is anAgrobacterium mediated double border plant transformation vectorcontaining a neomycin phosphotransferase selectable marker under thecontrol of a cauliflower mosaic virus 35S promoter, and a tedanalactamsynthase gene fused to an hsp70 leader under the control of a figwortmosaic virus promoter.

[0044]FIG. 24 represents a plasmid map of pMON25042 which contains alysine oxidase gene fused to a petunia hsp70 leader sequence under thecontrol of a figwort mosaic virus promoter.

[0045]FIG. 25 represents a plasmid map of pMON25050 which is anAgrobacterium mediated double border plant transformation vectorcontaining a neomycin phosphotransferase selectable marker under thecontrol of a cauliflower mosaic virus 35S promoter, and a lysine oxidasegene fused to a petunia hsp70 leader sequence under the control of afigwort mosaic virus promoter.

[0046]FIG. 26 represents a plasmid map of pMON33700 which is a planttransient expression vector containing a lysine oxidase gene fused to asequence encoding a maize ATP synthase beta subunit mitochondrialtransit peptide, which is fused to an hsp70 intron sequence under thecontrol of a root tissue enhanced promoter.

[0047]FIG. 27 represents a plasmid map of pMON33701 which is a planttransformation vector containing a neomycin phosphotransferase geneunder the control is of a cauliflower mosaic virus 35S promoter, and atedanalactam synthase gene fused to an hsp70 intron sequence under thecontrol of a root tissue enhanced promoter.

[0048]FIG. 28 represents a plasmid map of pMON33702 which is a planttransformation vector containing a neomycin phosphotransferase geneunder the control of a cauliflower mosaic virus 35S promoter; a lysineoxidase gene fused to a sequence encoding a maize ATP synthase betasubunit mitochondrial transit peptide, which is fused to an hsp70 intronsequence under the control of a root tissue enhanced promoter; and atedanalactam synthase gene fused to an hsp70 intron sequence under thecontrol of a root tissue enhanced promoter.

[0049]FIG. 29 represents a plasmid map of pMON38800, which is a planttransformation vector containing a neomycin phosphotransferase geneunder the control of a cauliflower mosaic virus 35S promoter; a lysineoxidase gene fused to an amino terminal His6 coding region, a ricemalate dehydrogenase amino terminal peroxisomal targeting signal, anintron and a 5′ wheat chloroplast AB untranslated leader under thecontrol of a 4AS1 promoter and a wheat 17 kd heat shock protein 3′untranslated sequence; and a tedanalactam synthase gene fused to anintron under the control of a 4AS1 promoter and a nopaline synthase 3′untranslated sequence.

DETAILED DESCRIPTION OF THE INVENTION

[0050] The following detailed description of the invention is providedto aid those skilled in the art in practicing the present invention.Even so, the following detailed description should not be construed tounduly limit the present invention as modifications and variations inthe embodiments discussed herein may be made by those of ordinary skillin the art without departing from the spirit or scope of the presentinventive discovery.

[0051] The work described herein has identified compositions and methodsof expressing an amino acid oxidase gene in combination with a secondgene encoding a protein which, when provided in a composition with theamino acid oxidase, confers plant resistance to coleopteran insects.Agronomic, horticultural, ornamental, and other economically orcommercially useful plants can be made in a functionally operablemanner, described herein, to express effective levels of protein toconfer resistance to coleopteran insects. Such plants may co-express thegenes encoding the amino acid oxidase and the second gene along withother genes encoding antifungal, antibacterial, or antiviralpathogenesis-related peptides, polypeptides, or proteins; insecticidalproteins; proteins conferring herbicide resistance; and genes encodingproteins involved in improving the quality of plant products oragronomic performance of plants. Simultaneous co-expression of multipleproteins in plants is advantageous in that it exploits more than onemode of action to control plant pathogenic damage. This can minimize thepossibility of developing resistant pathogen strains, broaden the scopeof resistance, and potentially result in a synergistic effect, therebyenhancing the level of resistance. Note WO 92/17591, for example, inthis regard. Examples are provided herein in which a lysine oxidase anda second gene which encodes a tedanalactam synthase are used incombination to control coleopteran insects through either direct feedingor by use of microbes expressing genes which encode these proteins.

[0052] As used herein, the term composition means a mixture ofingredients which, when combined, provides an insecticidally effectivesubstance containing an amount of the active ingredients encoded by afirst enzyme comprising an amino acid oxidase and a second enzyme thatprovides insecticidal activity when the second enzyme is present in themixture with the first enzyme. The composition can be an artificialmixture comprising the two active ingredients along with aqueous ornon-aqueous ingredients which may be combined to provide a substratesuitable for feeding to an insect. The feeding insect can be a larvaeform or may be an adult form. The composition can also be a mixturewhich contains one or more bacteria expressing the genes encoding thefirst and the second enzymes, such that the mixture, when provided in asuitable form for feeding to insects, causes an insecticidally effectiveamount of the two gene products to be delivered to the feeding insects.The composition can also be a mixture within plant tissues or plantcells, produced as a result of expression of the genes encoding thefirst and the second enzymes by the plant nuclear or nucleolar genome,by the plant mitochondrial genome, or by the plant chloroplast genome,such that the mixture, when consumed by a feeding insect, causes aninsecticidally effective amount of the two gene products to be deliveredto the feeding insects.

[0053] By “controlling insect infestation” it is meant that thecomposition upon which susceptible insects feed, usually a cropexpressing the contemplated genes, is capable of delivering aninsecticidally effective amount of the amino acid oxidase and the secondgene product to the feeding insect so that insect growth is stunted, orslowed, or that the insect dies without causing an unnecessary orunacceptable amount of crop damage.

[0054] “A plurality of cells” is intended to mean two or more cells, andthe cells can be of any type which are capable of being transformed withthe genetic constructs described herein, such as bacterial cells, plantcells, yeast cells, insect cells, and fungal cells.

[0055] “An insecticidally effective amount” is intended to mean anyamount of any composition described herein capable of providinginsecticidal activity upon ingestion of the composition by a susceptibleinsect, wherein the composition contains at least an amino acid oxidaseand a second gene product that provides insecticidal activity whenpresent in a mixture with the amino acid oxidase. The insecticidalactivity is readily observed upon ingestion by a susceptible insect. Asusceptible insect which consumes an insecticidally effective amountwill not continue to grow at the same rate as a control susceptibleinsect.

[0056] Stringent conditions as related to polynucleotide hybridizationis well known in the art, and so one skilled in the art would know thata number of factors are applicable, both in influencing the stability ofhybrid polynucleotide molecules and in influencing the hybridizationrate of polynucleotides. For example, factors which influence hybridstability include ionic strength of any hybridization solution; the basecomposition of the probe and the target polynucleotides; destabilizingagents present in the hybridization solution such as formamide or urea;the presence or availability of mismatched base pairs; and the duplexlength of the probe or target. All of these factors also influence thehybridization rate in addition to the temperature selected forhybridization; the viscosity of the solution; the complexity of theprobe, meaning the presence of repetitive sequences which would tend toincrease the hybridization rate; the pH of the hybridization solution;and the base composition of the probe and target. One skilled in the artwould be able to determine the optimum conditions for establishingstringency as related to identifying a polynucleotide by usinghybridization under stringent conditions to a probe of known base paircomposition.

[0057] Trichoderma sp. genes that encode 1) lysine oxidase (E.C.1.4.3.14) proenzyme and 2) a M_(r) 50,000 tedanalactam synthase havebeen isolated and sequenced. These new genes or genes from otherorganisms known to produce or capable of producing lysine or other aminoacid oxidases or proproteins and a tedanalactam synthase may be insertedinto expressible cassettes which can then be placed into atransformation vector for use in transforming plant-colonizingmicroorganisms which, when applied to plants, express the genesproducing these proteins, thereby providing control of insects.Alternatively, genes which are also part of expression cassettes, andwhich function in plants to encode the subject proteins may be insertedinto plant transformation vectors for use in transforming the genome ofa plant or plant subcellular organelle such as a mitochondria orchloroplast. Such transformed plants are thus provided with a capabilityto protect itself from attack by expressing the recombinant gene orgenes and producing a lysine or amino acid oxidase along with atedanalactam synthase. Additionally, the plant expressing thiscombination of insecticidal proteins may also be transformed or crossedwith other recombinant plants to obtain plants expressing B.t. genes forthe control of either the same or additional insects. Alternatively, therecombinant plants expressing this combination of insecticidal proteinsmay be crossed to other, more preferable germ plasm for purposes ofproviding commercial product lines. Examples of plants transformed toexpress B.t. genes are disclosed in European Patent Publication No. 0385962 (Fischhoff and Perlak, 1990).

[0058] An expression cassette is intended to mean a DNA moleculecomprising a promoter sequence which functions to cause the productionof an RNA sequence from a DNA sequence containing an open reading frameencoding either an amino acid oxidase, a second enzyme which providesinsecticidal activity when present in a mixture with an amino acidoxidase, or a chimeric gene open reading frame constructed from the inframe fusion of an amino acid oxidase and a second enzyme which providesinsecticidal activity when present in a mixture with an amino acidoxidase. The promoter sequence is required to be operably or operativelylinked to the DNA sequence containing one or more of the open readingframes. It is understood that the promoter chosen is functional in aparticular cell type. The cell type in which a promoter is functionalcan be any known in the art, in particular a bacterial or bacterialvirus promoter is known to be functional in various bacterial species; aplant or plant virus promoter is known to be functional in various plantspecies or in various plant cells, either tissue specific or non-tissuespecific. A promoter which is root enhanced or root specific or roottissue specific is one which provides expression preferentially in cellsderived from root tissues in a variety of plant types, and moreparticularly in corn, rice, wheat, soybean, canola, and cotton. By rootenhanced it is meant that the promoter has been isolated or geneticallyengineered to preferentially express or be more active in root tissuewhen compared to other plant tissue types such as leaves, fruit,flowers, and such. In eukaryotic cell types, and preferably in plantcell types, a particular expressible cassette includes a 3′non-translated DNA sequence which functions in plants to cause theaddition of a polyadenylated nucleotide sequence to the 3′ end of an RNAsequence generated from the function of an upstream operable promoterand gene sequence.

[0059] Expressible cassettes can also contain other elements in additionto those described above. For example, a DNA sequence which specifies a5′ non-translated leader sequence can be present downstream of thepromoter but upstream of the translatable gene sequence. DNA whichspecifies leader sequences are generally found in association withparticular promoter sequences, yet have been successfully utilized whenassociated with heterologous promoters and gene sequences. Such leadersequence can increase expression levels of desired proteins whenassociated with particular genes. Another DNA sequence which canincrease expression levels of desired proteins when associated withparticular genes specifies a non-translated RNA sequence known as anintron. Introns are well known in the art. Intron sequences are excisedpost-transcriptionally from the initial RNA transcript while stillwithin the boundaries of the nuclear envelope in a process known assplicing in which RNA 5′ adjacent to the intron sequence and 3′ adjacentto the intron sequence are fused to create the resulting mRNA sequencewhich is ultimately translated into a protein product. In thisapplication, intron sequences are preferred in gene constructs which areprepared for insertion into monocotyledenous species of plants, howeverthis is not meant to be limiting, because introns can function indicotyledenous species for the same purposes. Introns preferably areprovided downstream of a promoter but upstream of the gene codingsequence, however introns can also be placed within a gene codingsequence, or even downstream of a gene coding sequence but upstream of a3′ nontranslated polyadenylation coding sequence. Other elements can besignal peptide encoding sequences or transit peptide encoding sequences.These are also well known in the art. Signal peptides are ubiquitous andare generally fifteen to thirty amino acids long and are directed totargeting a precursor peptide, often a nascent peptide, to a secretionor secretory apparatus within the cell. The secretory apparatus can befound on or within the cytoplasmic or intracytoplasmic membrane of abacterium or the endoplasmic reticulum, Golgi, or other vacuolar orcytoplasmic membrane surface to which the signal peptide directs theprecursor peptide. The signal peptide is generally cleaved by somefaction of the secretory apparatus to release a proenzyme in which casethe precursor would be a pre-proenzyme, or to release a mature peptide.A targeting or transit peptide encoding sequence similarly directs aprotein to a particular membrane surface, which can be either a plastid,a chloroplast, or a mitochondria. The targeting or transit peptide leadsthe attached protein sequence into the particular organelle, and isgenerally cleaved to release the mature peptide into the particularorganelle.

[0060] A DNA vector can be any of a number of constructions which arewell known in the art. These can be selected from the group consistingof but not limited to plasmid, bacmid, phage, cosmid, yeast artificialchromosome (YAC), bacterial artificial chromosome (BAC), plant virus, orlinear DNA or RNA, so long as the vector is capable of being deliveredto a target cell for the express purpose of either transforming the cellby incorporation into the genome of the cell, by stable or temporaryreplication or otherwise existing for a period of time within the targetcell so that the genes encoding proteins, which in combination provideor exhibit coleopteran insecticidal activity, are able to produce thecontemplated composition either for purposes of enzymatic orimmunological detection or for protection of a plant or plant cell frominsect damage.

[0061] The present invention includes not only the Trichoderma lysineoxidase and tedanalactam synthase proteins, but also biologicallyequivalent peptides, polypeptides, and proteins. The phrase“biologically equivalent peptides, polypeptides, and proteins” denotespeptides, polypeptides, and proteins that exhibit the same or similaractivities when assayed in comparison to the Trichoderma counterpart byin vitro or in vivo assays. The phrase “same or similar activities”denotes the ability to perform the same or similar function as theTrichoderma counterpart. These peptides, polypeptides, and proteins cancontain a region or moiety exhibiting sequence similarity to acorresponding region or moiety of the Trichoderma proteins disclosedherein, but this is not required as long as they exhibit the same orsimilar activity as their Trichoderma counterpart. Biologicallyequivalent peptides, polypeptides, and proteins may include, but are notlimited to truncated fragments deleted from the N-terminal end,C-terminal end, internal regions of the protein, or combinationsthereof. Additionally, variants resulting from changes in one or moreamino acid to a different natural or non-natural amino acid, deletions,or insertions of natural or non-natural amino acids may result in abiologically equivalent compound. Such variants may be naturallyoccurring materials, or may be produced by mutagenesis or randommutagenesis of the encoding nucleotide sequence.

[0062] The present invention encompasses not only the Trichoderma DNAsequences listed, but also biologically functional equivalent nucleotidesequences. The phrase “biologically functional equivalent nucleotidesequences” denotes DNAs and RNAs, including genomic DNA, cDNA, syntheticDNA, and mRNA nucleotide sequences, that encode peptides, polypeptides,and proteins exhibiting the same or similar activities as theTrichoderma lysine oxidase or tedanalactam synthase when assayed by invitro or in vivo methods. Such biologically functional equivalentnucleotide sequences can encode peptides, polypeptides, or proteins thatcontain a region or moiety exhibiting sequence similarity to thecorresponding region or moiety of the Trichoderma counterpart.Nucleotide sequences may contain conservative amino acid changes,altering the codon usage for a particular amino acid, but leaving theencoded peptide, polypeptide, or protein sequence unchanged.Alternatively, nucleotide sequences may contain non-conservative changesincluding, but not limited to substitutions, deletions, additions, orcombinations thereof. These biologically functional equivalentnucleotide sequences may be naturally occurring or produced by in vitromethods. These biologically functional equivalent nucleotide sequencesare preferably 80% identical to their Trichoderma counterparts. Morepreferably, biologically functional equivalent nucleotide sequences are85%, 87.5%, 90%, 92.5%, 95%, 97.5%, and ideally 100% identical to theirTrichoderma counterparts. Biologically functional equivalent nucleotidesequences may be identified by their capability of hybridizing understringent conditions to the lysine oxidase or tedanalactam synthaseencoding sequences, or the complements thereof.

[0063] Methods for identifying other genes which encode a first geneencoding an amino acid oxidases and a second gene which encodes aprotein that provides insecticidal activity when present in a mixturewith an amino acid oxidase are contemplated herein. Methods foridentifying such sequences are well known in the art, however thenovelty of the second gene provides a particular advantage to theinvention. The second gene described herein as a tedanalactam synthasecan be used to detect and identify the presence of other genes ofsubstantial similarity, meaning genes which are capable of beingdetected by hybridization to the tedanalactam synthase gene. Any mixtureor sample containing a polynucleotide sequence encoding a protein thatprovides insectidical activity when present in a mixture with an aminoacid oxidase can be probed with a labeled polynucleotide sequence whichis or is complementary to all or a portion of the tedanalactam synthasegene. The act of probing a sample with the tedanalactam synthase genewill provide a probe/sample complex in mixtures which contain ahomologous gene or a heterologous gene capable of binding to the probe.The probe/sample complex can be detected in any number of ways notlimited to enhanced chemiluminescence, radioisotopic, fluorescent, orcolorimetric methods well known in the art. The complex can be isolated,particularly if the method chosen has utilized a phage blot or a cellculture blot method. The polynucleotide or polynucleotides which boundto the probe can be isolated either from the probe/sample complex orderived from the particular sample which gave rise to the probe/samplecomplex to yield a gene which encodes a protein that providesinsectidical activity when present in a mixture with an amino acidoxidase. The gene isolated in this way may or may not have tedanalactamsynthase activity.

[0064] Antibodies can be generated to detect either of the activepeptides which comprise the compositions herein. An amino acid oxidaseenzyme or a tedanalactam synthase enzyme can be purified by any numberof means well known in the art. Purified enzymes can be provided inadjuvant form for injection into a variety of animals, also well knownin the art. Preferably rabbits are used, however goats, guinea pigs,horses, turkeys, chickens, and even humans could be used for producingreagent grade antiserum directed to particular epitopes of theseproteins for use in methods which utilize antibodies for detection andpurification of such peptides. While serum from animals providespolyclonal antibodies which are directed to a large number or a varietyof epitopes on each protein, monoclonal antibodies could easily beproduced by methods well known in the art.

[0065] Kits could also be used, in particular when immunologicalreagents are available, such as antibodies directed to detection ofamino acid oxidase or tedanalactam synthase enzyme, or by designingoligonucleotide sequences for use in detecting the presence of genescontemplated herein by thermal amplification methods or in combinationwith immunological methods.

[0066] The expression of a plant gene which exists in double-strandedDNA form involves transcription of messenger RNA (mRNA) from one strandof the DNA by RNA polymerase enzyme, and the subsequent processing ofthe mRNA primary transcript inside the nucleus. This processing involvesa 3′ non-translated region which adds polyadenylate nucleotides to the3′ end of the RNA. Transcription of DNA into mRNA is regulated by aregion of DNA usually referred to as the “promoter”. The promoter regioncontains a sequence of bases that signals RNA polymerase to associatewith the DNA and to initiate the transcription of mRNA using one of theDNA strands as a template to make a corresponding strand of RNA.

[0067] One skilled in the art will recognize that many promoters, 5′non-translated leader sequences, introns and polyadenylation sequencesmay be used in accordance with the present invention. Suitable examplesof promoter sequences include, but are not limited to nopaline synthase(NOS), octopine synthase (OCS), cauliflower mosaic virus 19S and 35S(CaMV19S, CaMV35S), ribulose 1,5-bisphosphate carboxylase (ssRUBISCO),figwort mosaic virus (FMV), asparagine synthase,glutathione-S-transferase, T-DNA, CPRF1, histone H3, wheat gliadin,nopaline synthase, Agrobacterium rhizogenes rolC, tobacco anionicperoxidase, napA storage protein, Cassava vein mosaic virus (CVMV),polyubiquitin, glycinin Gy2, mas, mustard CHS1, Chlorella virus adeninemethyltransferase, Arabidopsis phenylalanine ammonia-lyase, potato ubi3,and the tomato hmg2 promoter. Suitable examples of root enhanced or rootspecific promoter sequences include, but are not limited to CaMV derivedAS4, tobacco RB7, and the rice RC2 promoter. Suitable examples of intronsequences include, but are not limited to the maize heat shock protein70 (hsp70), rice actin, cox11, histone H3, RNA polymerase II,chloroplast DNA trnl, maize ADH1 intron 1, maize actin intron 3,Arabidopsis thaliana polyubiquitin, and the plant hemoglobin exon 2intron. Suitable examples of 5′ leader sequences include, but are notlimited to petunia heat shock protein 70 (hsp70), AMV RNA4, 16Sribosomal, Arabidopsis ACT2, Arabidopsis ACT8, TMV RNA, and soybean Gy2.Suitable examples of polyadenylation signals include, but are notlimited to Agrobacterium nopaline synthase (NOS) and the Pisum sativumRUBISCO small subunit E9.

[0068] A number of promoters which are active in plant cells have beendescribed in the literature. Such promoters may be obtained from plantsor plant viruses and include, but are not limited to, the nopalinesynthase (NOS) and octopine synthase (OCS) promoters (which are carriedon tumor-inducing plasmids of Agrobacterium tumefaciens), thecauliflower mosaic virus (CaMV) 19S and 35S promoters, thelight-inducible promoter from the small subunit of ribulose1,5-bisphosphate carboxylase (ssRUBISCO, a very abundant plantpolypeptide), and the Figwort Mosaic Virus (FMV) 35S promoter. All ofthese promoters have been used to create various types of DNA constructswhich have been expressed in plants (see e.g., Barry and Kishore, U.S.Pat. No. 5,463,175).

[0069] The particular promoter selected should be capable of causingsufficient expression of the enzyme coding sequence to result in theproduction of an insecticidal effective amount of lysine oxidase andtedanalactam synthase. One set of preferred promoters are constitutivepromoters such as the CaMV35S or FMV35S promoters that yield high levelsof expression in most plant organs. Another set of preferred promotersare root enhanced or specific promoters such as the CaMV derived AS4promoter, the tobacco RB7 promoter, or the rice RC2 promoter (Lam etal., 1991; Yamamoto et al., 1991; Hertig et al., 1991; Xu et al., 1995).The root enhanced or specific promoters would be particularly preferredfor the control of corn rootworm (Diabrotica spp.) in transgenic cornplants.

[0070] The promoters used in the DNA constructs (i.e. chimeric plantgenes) of the present invention may be modified, if desired, to affecttheir control characteristics. For example, the CaMV35S promoter may beligated to the portion of the ssRUBISCO gene that represses theexpression of ssRUBISCO in the absence of light, to create a promoterwhich is active in leaves but not in roots. The resulting chimericpromoter may be used as described herein. For purposes of thisdescription, the phrase “CaMV35S” promoter thus includes variations ofCaMV35S promoter, e.g., promoters derived by means of ligation withoperator regions, random or controlled mutagenesis, etcetera.Furthermore, the promoters may be altered to contain multiple “enhancersequences” to assist in elevating gene expression. Examples of suchenhancer sequences have been reported by Kay et al. (1987).

[0071] The RNA produced by a DNA construct of the present invention alsocontains a 5′ non-translated leader sequence. This sequence can bederived from the promoter selected to express the gene, and can bespecifically modified so as to increase translation of the mRNA. The 5′non-translated regions can also be obtained from viral RNAs, fromsuitable eucaryotic genes, or from a synthetic gene sequence. Thepresent invention is not limited to constructs wherein thenon-translated region is derived from the 5′ non-translated sequencethat accompanies the promoter sequence. As shown below, a plant geneleader sequence which is useful in the present invention is the petuniaheat shock protein 70 (hsp70) leader (Winter et al., 1988).

[0072] For optimized expression in monocotyledenous plants, an intronshould also be included in the DNA expression construct. This intronwould typically be placed near the 5′ end of the mRNA in untranslatedsequence. This intron could be obtained from, but not limited to, a setof introns consisting of the maize hsp70 intron (Brown and Santino U.S.Pat. No. 5,424,412; 1995) or the rice Act1 intron (McElroy et al.,1990). As shown below, the maize hsp70 intron is useful in the presentinvention.

[0073] As noted above, the 3′ non-translated region of the chimericplant genes of the present invention contains a polyadenylation signalwhich functions in plants to cause the addition of adenylate nucleotidesto the 3′ end of the RNA. Examples of preferred 3′ regions are (1) the3′ transcribed, non-translated regions containing the polyadenylatesignal of Agrobacterium tumor-inducing (Ti) plasmid genes, such as thenopaline synthase (NOS) gene and (2) plant genes such as the peassRUBISCO E9 gene (Fischhoff et al., 1987).

[0074] A chimeric plant gene containing the structural coding sequencesof the present invention can be inserted into the genome of a plant byany suitable method. Suitable plant transformation vectors include thosederived from a Ti plasmid of Agrobacterium tumefaciens, as well as thosedisclosed, e.g., by Herrera-Estrella (1983), Bevan (1983), Klee (1985)and EPO publication 0 120 516 (Schilperoort et al.). In addition toplant transformation vectors derived from the Ti or root-inducing (Ri)plasmids of Agrobacterium, alternative methods can be used to insert theDNA constructs of this invention into plant cells. Such methods mayinvolve, for example, the use of liposomes, electroporation, chemicalsthat increase free DNA uptake, free DNA delivery via microprojectilebombardment, and transformation using viruses or pollen (Fromm et al.,1986; Armstrong et al., 1990; Fromm et al., 1990).

[0075] To identify a transgenic plant expressing lysine oxidase and/ortedanalactam synthase, it is necessary to screen the herbicide orantibiotic resistant transgenic, regenerated plants (R0 generation) forexpression of these genes. This can be accomplished by various methodswell known to those skilled in the art, including but not limited to: 1)obtaining small tissue samples from the transgenic R0 plant and directlyassaying the tissue for activity against susceptible insects in parallelwith tissue derived from a non-expressing, negative control plant. Forexample, R0 transgenic potato plants expressing lysine oxidase andtedanalactam synthase can be identified by assaying leaf tissue derivedfrom such plants for activity against CPB; 2) analysis of proteinextracts by enzyme linked immunoassays (ELISAs) or immunoblot assaysspecific for lysine oxidase and/or tedanalactam synthase (antibodiesuseful for such detection schemes are described in the examples) or 3)reverse transcriptase PCR (RT PCR) to identify events expressing thegene of interest.

[0076] The following examples describe preferred embodiments of theinvention. Other embodiments within the scope of the claims herein willbe apparent to one skilled in the art from consideration of thespecification or practice of the invention as disclosed herein. It isintended that the specification, together with the examples, beconsidered exemplary only, with the scope and spirit of the inventionbeing indicated by the claims which follow the examples. In the examplesall percentages are given on a weight basis unless otherwise indicated.

EXAMPLES

[0077] The present invention can be better understood from the followingillustrative, non-limiting Examples. Effective control of coleopteraninsect pests is demonstrated when these proteins are obtained orexpressed within their natural source organism, in heterologousmicroorganisms, or in transgenic plants.

Example 1

[0078] This example illustrates the discovery and characterization ofinsecticidal activity from a Trichoderma species.

[0079] The culture filtrate from a Trichoderma species, Monsanto fungalisolate # F22844, was found to exhibit insecticidal activity in asouthern corn rootworm bioassay. The proteinaceous nature of the activecomponent was suggested by characterization experiments which showedthat first, the corn rootworm activity was completely lost after heatingand second, only components within the filtrate which were larger than10 kDa in size maintained insecticidal bioactivity (Table 1). Aqualitative visual assessment of the assay revealed that the >10 kDasample severely stunted the surviving larvae and also had ovicidaleffects in addition to the mortality noted in Table 1.

[0080] A sample of a >10 kDa preparation of F22844 was utilized indiet-preincubation studies to determine if the corn rootworm activitywas due to a modification of the artificial diet. It was demonstratedthat diet pre-incubated with F22844 retained full insecticidal activity,while similar samples subjected to heat treatment prior to larvaladdition exhibited reduced bioactivity (Table 1).

[0081] An important consideration in determining the utility of aninsecticidal lead is bioactivity in bioassays using plant tissue in theassay media. F22844 retained its insecticidal activity in assays withplant tissue when a >10 kDa preparation of F22844 was added to BMS corncallus. Significant mortality of corn rootworm larvae feeding on thismaterial was seen in this bioassay (Table 1). TABLE 1 % Mortality Sizingand heat lability study [> 10 kDa] 68 [< 10 kDa] 0 [> 10 kDa] - Heated100° C. 0 Diet pre-incubation study Un-incubated 94 Incubated 94Incubated/Heated 80° C. 19 Callus diet assay [> 10 kDa] 94

Example 2

[0082] This example illustrated the identification and characterizationof proteins with insecticidal activity isolated from Trichodermafermentation extracts.

[0083] SDS-PAGE analysis of chromatography fractions generated duringthe purification of the southern corn rootworm-active protein(s) fromF22844 showed that major proteins of M_(r) 56,000 and M_(r) 50,000 werepresent in the corn rootworm-active fractions. These proteins were thenpurified by sizing and ion exchange chromatography. This protocolconsistently yielded significantly purified proteins. The bioassayresults obtained with purified proteins from two separate fermentationsof F22844 are shown in Tables 2a and 2b. In both cases, no bioactivitywas detected when the proteins were assayed individually but significantstunting was seen when the proteins were combined in assay. Theseresults demonstrate that it is a combination of the two proteins that isresponsible for the corn rootworm activity in F22844. TABLE 2a. Meanlarval weight Sample Conc. (μg/mL) (mg) ± (SEM) % Stunting Acetatebuffer 0 1.14 ± (0.17) — M_(r) 56,000 10 0.91 ± (0.09) NSS M_(r) 50,0001.5 0.83 ± (0.05) NSS M_(r) 56,000 + 10 + 1.5 0.37 ± (0.07) 67 M_(r)50,000

[0084] TABLE 2b Mean larval weight Sample Conc. (μg/mL) (mg) ± (SEM) %Stunting Acetate buffer — 1.29 ± (0.16) — M_(r) 56,000 2 1.63 ± (0.22) —M_(r) 50,000 2 1.39 ± (0.22) — M_(r) 56,000 + 2 + 2 0.56 ± (0.22) 57M_(r) 50,000

[0085] Native-PAGE studies were effective in confirming that the M_(r)56,000 and M_(r) 50,000 proteins were responsible for the southern cornrootworm bioactivity of F22844. The chromatographically purified M_(r)56,000 and M_(r) 50,000 proteins were further purified by native PAGE.SDS-PAGE analysis demonstrated that the proteins were purified to singlebands. The individual purified proteins did not yield statisticallysignificant stunting of corn rootworm (Table 3). A sample containing acombination of the two proteins at concentrations of 9.7 μg/mL of M_(r)56,000 and 2.5 μg/ml of M_(r) 50,000 yielded 89% stunting of cornrootworm larvae (Table 3). TABLE 3 Mean larval weight Sample Conc.(μg/mL) (mg) ± (SEM) % Stunting Acetate buffer 0 0.88 ± (0.11) — M_(r)56,000 9.7 0.84 ± (0.06) NSS M_(r) 50,000 2.5 1.04 ± (0.11) NSS M_(r)56,000 ± 9.7 ± 2.5 0.10 ± (0.01) 89 M_(r) 50,000

[0086] An SDS-PAGE gel of the proteins produced by Trichoderma F22844,isolated as above, were blotted onto a PVDF membrane (Immobilon,Millipore, Bedford, Mass.) using the protocol of Matsudaira (Matsudaira,1987). The N-terminus was sequenced using automated Edman degradationchemistry. A gas phase sequencer (Applied Biosystems, Foster City,Calif.) was used for the degradation using the standard sequencer cycle.The respective PTH-aa derivatives were identified by reverse phase HPLCanalysis in an on-line fashion employing a PTH analyzer (AppliedBiosystems, Foster City, Calif.) fitted with a Brownlee 2.1 mm i.d.PTH-C18 column. For internal sequences, digestions were carried out onpurified lysine oxidase and M_(r) 50,000 proteins from F22844 usingtrypsin (TPCK-treated, from Worthington Biochemicals Corp., Freehold,N.J.). Fragments were then purified by reverse phase HPLC and sequencedin an N-terminal fashion.

[0087] F22844 was identified taxonomically as a Trichoderma species. Asearch in the CRC antibiotic database for proteins produced byTrichoderma yielded L-lysine oxidase (LO), an M_(r) 56,000 proteinidentified as an antitumor agent (Kusakabe et al., 1979; 1980). Thepurified M_(r) 56,000 protein from F22844 was identified as L-lysineoxidase by enzymatic assay (Gallo et al., 1981). We determined thelysine oxidase from F22844 to have a K_(m) for lysine of 26 μM, a valuevery similar to the reported K_(m) for lysine of 40 μM for the T. viridelysine oxidase (Kusakabe et al., 1980). The substrate specificity ofF22844 lysine oxidase was also determined (Table 4). The relativereaction rates are very similar to the values in the literature for T.viride lysine oxidase (Kusakabe et al., 1980). TABLE 4 Relative activity(%) Substrate^(a) F22844^(a) T. viride ^(b) Lysine 100.0 100 Ornithine10.8 18.2 Phenylalanine 6.6 8.3 Histidine 5.0 3.8 Arginine 2.6 6.1

[0088] Data generated thus far indicate that the M_(r) 50,000 proteincatalyses the conversion of Δ¹-piperideine-2-carboxylate (P2C) toproduct I. High resolution mass spectral data suggest that I is the3,4-epoxide of 2-piperidone, which has previously been identified astedanalactam (Cronan and Cardellina, 1994). Hydrogen peroxide appears tobe the byproduct of the M_(r) 50,000 protein reaction. The formation ofhydrogen peroxide was monitored by a colorimetric assay (Gallo, 1981).P2C was prepared by two separate methods for the initial M_(r) 50,000protein assay. The two methods used for preparing P2C were

[0089] 1) L-lysine with lysine oxidase and catalase; and

[0090] 2) DL-pipecolic acid with D-amino acid oxidase and catalase.

[0091] P2C was isolated from the protein mixture by filtration using anAmicon filter (10 kDa cutoff) before using in the M_(r) 50,000 proteinassay. Pipecolic acid and lysine are not substrates of M_(r) 50,000protein.

[0092] Note that in proposing an enzymatic activity for the M_(r) 50,000protein, we do not limit our claims. More specifically, we propose thatboth the M_(r) 50,000 protein and any substantially homologous proteinswill yield coleopteran insect control when combined with lysine oxidaseand other amino acid oxidases. Moreover, it is conceived that any enzymecapable of converting Δ¹-piperideine-2-carboxylate to tedanalactam wouldbe considered by one skilled in the art to have tedanalactam syntheticactivity, and would be characterized as a tedanalactam synthase enzyme.Any tedanalactam synthase enzyme capable of convertingΔ¹-piperideine-2-carboxylate to tedanalactam could be used incompositions with lysine oxidase or other amino acid oxidases asdescribed herein for controlling insect infestation of plants, and mayalso be effective in controlling insect infestation in general whensupplied in any form capable of being ingested by an insect. Genesencoding tedanalactam synthases may not necessarily be homologous togenes encoding such enzymes isolated from fungal species such asTrichoderma. Other tedanalactam synthase genes may encode enzymes whichwould be substantially smaller than such enzymes observed fromTrichoderma. Conversely, it is entirely possible that still othertedanalactam synthase genes may encode enzymes which are substantiallylarger than such Trichoderma enzymes. Therefore, it is not desired thatthis invention be limited to enzymes approximately M_(r) 50,000 havingtedanalactam synthase activity.

Example 3

[0093] This example illustrates the bioactivity of lysine oxidase andthe tedanalactam synthase derived from naturally occurring sourceorganisms.

[0094] Lysine oxidase and tedanalactam synthase were purified fromculture filtrates of the native F22844 fungus. Both proteins weregreater than 90% pure by SDS-PAGE. Concentration response curves wererun to determine the efficacy of these purified proteins on neonatewestern corn rootworm, Diabrotica virgifera virgifera LeConte larvae. AnLC₅₀ below 2 ppm of each protein was demonstrated (Table 5). TABLE 5Sample Conc. (ppm) % Mortality % Stunting LO + M_(r) 50,000 20 + 20 100— 10 + 10 100 — 2 + 2 63 80 Control — 0  0

[0095] Western corn rootworm, Diabrotica virgifera virgifera LeConte,bioassays were also conducted using second instar larvae with lysineoxidase present at 30 ppm and tedanalactam synthase present at 4 ppm. Byday 10, the western corn rootworm larvae exposed to the active proteinshad a corrected mortality of 81% and the surviving larvae actually lostweight over the course of the assay (Table 6). TABLE 6 Larval Larval#Larv. Wts. (mg) - #Larv. #Larv. #Larv. #Larv. Wts. (mg) - Initial Day 1± Surv. Surv. Surv. Surv. Day 10 ± Sample Day 1 (SEM) Day 4 Day 7 Day 9Day 10 (SEM) Buffer 48 2.63 ± 46 44 43 43 8.48 ± control (0.18) (0.57)Lysine 48 2.63 ± 38 21 10 8 1.44 ± oxidase + (0.18) (0.10) M_(r) 50,000

[0096] Concentration response studies using chromatographically purifiedproteins were conducted. A concentration of 10 ppm of each protein(lysine oxidase+tedanalactam synthase) yielded 80% stunting of southerncorn rootworm while 2 ppm of each yielded 60% stunting (Table 7). TABLE7 Mean larval weight % Sample Conc. (μg/mL) (SEM) Stunting Acetatebuffer 0 1.00 (0.11) — Lysine oxidase + 2 + 2 0.40 (0.05) 60 M_(r)50,000 Lysine oxidase + 10 +10 0.20 (0.05) 80 M_(r) 50,000

[0097] The purified proteins were also bioassayed in a BMS callus dietand bioactivity was retained in this assay (Table 8). These datademonstrated that the proteins were bioactive against southern cornrootworm in plant based bioassays in addition to artificial dietbioassays. TABLE 8 Mean larval weight % Sample Conc. (μg/mL) (mg) ±(SEM) Stunting Artificial Diet Acetate buffer 0 1.15 ± (0.28) — Lysineoxidase + 17 + 2 0.35 ± (0.06) 70 M_(r) 50,000 BMS Callus Acetate buffer0 0.27 ± (0.04) — Lysine oxidase + 17 + 2 0.13 ± (0.01) 53 M_(r) 50,000

[0098] Bioassays were done with purified M_(r) 56,000+M_(r) 50,000proteins from F22844 against two other coleopteran insects. Bioactivitywas detected against Colorado potato beetle, Leptinotarsa decemlineata(Say), and boll weevil, Anthonomus grandis grandis Boheman. TABLE 9 MeanLarval wt Sample Surv/Init % Mortality (mg) ± (SEM) % Stunting Bollweevil Buffer control 30/32 — 11.05 ± (1.49)  — LO + 50 (2 ppm each)13/16 13 12.07 ± (2.05)  — LO + 50 (6 ppm each) 11/16 27 5.67 ± (2.10)49 LO + 50 (18 ppm each)  6/16 60 1.42 ± (0.48) 87 CO. potato beetleBuffer control 31/32 — 3.31 ± (0.33) — LO + 50 (2 ppm each) 15/16  32.83 ± (0.24) 15 LO + 50 (6 ppm each) 12/16 22 2.57 ± (0.48) 23 LO + 50(18 ppm each)  8/16 48 2.08 ± (0.32) 37

[0099] Culture supernatants from four other Monsanto fungal isolatesthat had corn rootworm insecticidal activity were strongly positive forlysine oxidase enzymatic activity—F25528, F25634, F26040 and F25508.These leads were not characterized further. Culture supernatants fromATCC T. viride isolates #20536 and #20538 expressed lysine oxidase andan M_(r) 50,000 protein and were insecticidally active against southerncorn rootworm. Lysine oxidase from Trichoderma viride is availablecommercially from Sigma (St. Louis, Mo.). The enzyme was purified to asingle band from this preparation and bioassayed against southern cornrootworm. Bioassays were conducted to compare the concentrationresponses of the T. viride lysine oxidase (±M_(r) 50,000 protein fromF22844) with the lysine oxidase purified from F22844. The bioactivity ofthe T. viride lysine oxidase agrees very well with the bioactivity oflysine oxidase from F22844 when bioassayed in the presence oftedanalactam synthase from F22844 (Table 10). Very similar stuntinglevels are seen at equivalent doses (compare Table 10 to Table 7). Inaddition, no bioactivity is seen with either lysine oxidase in theabsence of tedanalactam synthase from F22844. TABLE 10 Mean larvalweight Sample Conc. (ppm) (mg) ±](SEM) % Stunting Buffer control 0 0.77± (0.09) — T. viride LO 10  0.74 ± (0.07) 4* T. viride LO 2 0.73 ±(0.09) 5* T. viride LO + 10 + 10 0.21 ± (0.04) 73 F22844 M_(r) 50,000 T.viride LO + 2 + 2 0.39 ± (0.05) 49 F22844 M_(r) 50,000

Example 4

[0100] This example illustrates mode of action studies of the lysineoxidase and tedanalactam synthase and effects which were observed on thecorn root worm midgut.

[0101] The effects of lysine oxidase and tedanalactam synthase proteinson the morphology and ultrastructure of the southern corn rootwormmidgut were investigated by light and electron microscopy. Lightmicroscopy showed that the midguts were intact with microvilli but therewas marked apical folding of epithelium and basal infolding in treatedindividuals and an apparent loss of the basal regenerative cells.Several ultrastructural changes were evident by electron microscopy. Therough endoplasmic reticulum of the epithelial cells was dramaticallyreduced in treated individuals indicating a reduced potential forprotein synthesis. There was an increased electron density (osmiophilia)of lateral plasma membranes suggesting an abnormality in lipidmetabolism. In the fat body, lipid vesicles were significantly reducedin treated individuals. These micrographs showed that there are somedefinite cellular changes associated with the treatment of corn rootwormwith the purified proteins.

Example 5

[0102] This example describes the isolation and characterization of thegenes encoding a lysine oxidase and a tedanalactam synthase.

[0103] The lysine oxidase and tedanalactam synthase genes were isolatedfrom one of the Trichoderma sp. microorganisms isolated in Ecuador andthe sequences determined.

[0104] Peptide sequences from purified tryptic peptides from F22844lysine oxidase were obtained and used to design degenerateoligonucleotides to clone the lysine oxidase gene. From peptide P1 (SEQID NO: 1) was deduced the antisense strand oligonucleotide N1 (SEQ IDNO: 2), from peptide P2 (SEQ ID NO: 3) was deduced the antisense strandoligonucleotide N2 (SEQ ID NO: 4) and sense strand oligonucleotides N3,N4, and N5 (SEQ ID NOS:5,6,7). From peptide P3 (SEQ ID NO: 8) wasderived the sense strand oligonucleotides N6 (SEQ ID NO: 9) and N7 (SEQID NO: 10). P1 (SEQ ID NO:1):Asp Ala Pro Pro Gin Pro Pro Lys Glu Asp Glu Leu Val Glu Leu IleLeu Gin Asn Leu Ala Arg N1 (SEQ ID NO:2):TC(AG) TC(CT) TC(CT) TTI GGI GG(CT) TG P2 (SEQ ID NO:3):Gly Leu Asn Leu His Pro Thr Gln Ala Asp Ala Ile Arg N2 (SEQ ID NO:4):ATIGC(AG)TCIGC(CT)TGIGTIGG(AG)TG N3 (SEQ ID NO:5):CA(CT)CCIACICA(AG)GCIGA(CT)GCIAT N4 (SEQ ID NO:6):AA(CT)CTICA(CT)CCIACICA(AG)GC N5 (SEQ ID NO:7):AA(CT) TT(AG) CA(CT) CCI ACI CA(AG) GC P3 (SEQ ID NO:8):Lys Gln Gln Ala Phe Gly Tyr Tyr Lys N6 (SEQ ID NO:9):AA(AG)CA(AG)CA(AG)GCITT(CT)GGITA N7 (SEQ ID NO:1O):CA(AG)GCITT(CT)GGITA(CT)TA(CT)AA

[0105] To obtain a partial cDNA clone, nested sense and antisensedegenerate oligonucleotides to three internal tryptic peptide sequencesfrom the purified lysine oxidase were randomly combined in separatepair-wise PCR reactions with total cDNA from Trichoderma F22844 (Lee atal, 1988). The combination of sense strand oligonucleotides from peptideP3 (SEQ ID NO: 8) and an antisense strand oligonucleotide from peptideP1 (SEQ ID NO: 1) yield a PCR product of approximately 800 bprepresenting an internal portion of the lysine oxidase cDNA. Morespecifically, sense strand oligonucleotides N6 (SEQ ID NO: 9) or N7 (SEQID NO: 10) were combined with the antisense strand oligonucleotide N1(SEQ ID NO: 2) in a 50 μL PCR reaction containing 50 picomoles of eacholigonucleotide, 1× TAQ buffer (Perkin-Elmer, Norwalk, Conn.), 1.5 mMMgCl₂, 660 μM dNTPs, and 0.5 units of Taq polymerase. The thermocyclingprofile was 1 cycle at 94° C. 5 minutes, 80° C. for 5 minutes in theabsence of Taq polymerase followed by addition of Taq. After Taqaddition, there were 3 cycles of 94° C. for 1 minute, 30 second ramp to37° C. for 30 seconds, a 2.5 minute ramp to 72° C. for 2 minutes. Thiswas followed by a PCR profile of 37 cycles of 94° C. for 1 minute, 48°C. for 1 minute, 72° C. for 2 minutes and terminated with a finalincubation at 74° C. for 4 minutes.

[0106] To clone the internal lysine oxidase cDNA fragment, the 800 bpPCR fragment can then be ligated as a blunt ended fragment into acloning vector such as pBSIIKS+ (Stratagene, La Jolla, Calif.) digestedwith SmaI to produce plasmid pMON23671 (FIG. 3). DNA sequencing of thisfragment reveals DNA sequences coding for peptide fragments P2 (SEQ IDNO: 3) and P4 (SEQ ID NO: 11) as well as portions of peptide fragmentsP1 (SEQ ID NO: 1) and P3 (SEQ ID NO: 8), confirming the identity of theclone as a lysine oxidase cDNA fragment (SEQ ID NO: 12). Note that thefirst residue of the P3 (SEQ ID NO: 8) was later found to be a leucinerather than a lysine upon review of both the DNA and protein sequencingdata. The oligonucleotide N7 (SEQ ID NO: 10) apparently still hybridizedand functioned under the conditions described since only the first twonucleotides at its 5 prime end are mismatched.

[0107] It is useful to identify a complete cDNA sequence to determine ifthe genomic DNA contains introns that disrupt the coding sequence. Suchintrons could inhibit expression of the cloned gene in heterologoussystems such as plants. To obtain the complete sequence of the lysineoxidase cDNA, the standard RACE (Rapid Amplification of cDNA Ends)procedure (Frohman et al., 1988) was used to extend the cDNA sequencefrom the internal core cDNA sequence present in pMON23671.

[0108] To recover the 3′ end, cDNA synthesized from poly A enrichedF22844 with the 3′ Race Adapter primer (Gibco-BRL, Gaithersburg, Md.)was PCR amplified with the Universal Amplification Primer (Gibco-BRL,Gaithersburg, Md.) and the lysine oxidase sense strand oligonucleotideN8 (SEQ ID NO: 13). This PCR reaction yields a product of approximately700 bp that can be re-amplified with the internally nestedoligonucleotide N9 (SEQ ID NO: 14) to yield a PCR product ofapproximately 650 bp. The 650 bp product was subsequently subcloned as ablunt ended fragment into EcoRV digested pBSIIKS+ to yield pMON23683(FIG. 4). Subsequent sequencing of this cDNA clone displayeduninterrupted homology to the genomic lysine oxidase genomic DNAsequence (SEQ ID NO: 15). N8 (SEQ ID NO:13): ACCTCTACGAACTTGCGTTTACC N9(SEQ ID NO:14): CAACTCGCATTGGATCGTTGGTG

[0109] To recover the 5′ end, two sets of 5′ RACE reactions wereperformed. In the first set, poly A enriched RNA from F22844 was reversetranscribed into cDNA with oligonucleotide N10 (SEQ ID NO: 16) and dCtailed with Terminal Transferase (Gibco-BRL, Gaithersburg, Md.). ThiscDNA was then amplified first with oligonucleotide N11 (SEQ ID NO: 17)and the Anchor Primer or AP (Gibco-BRL, Gaithersburg, Md.). This PCRreaction was subsequently re-amplified with oligonucleotide N12 (SEQ IDNO: 18) and the Universal Amplification Primer or UAP (Gibco-BRL,Gaithersburg, Md.) to yield a 300 bp product which was blunt end clonedinto EcoRV digested pBSIIKS+ to yield pMON23681 (FIG. 5). Sequenceanalysis of this cDNA clone (SEQ ID NO: 19) revealed uninterruptedhomology to the genomic lysine oxidase genomic DNA sequence. However, asecond set of 5′ RACE reactions was then needed to recover the remaining5′ portion of the lysine oxidase cDNA sequence. This was accomplished byuse of oligo dT primed first strand cDNA as template, followed by oneround of PCR amplification with the AP and N13 (SEQ ID NO: 20), andcompleted with a final round of PCR amplification using the precedingPCR reaction product as template with the UAP and N14 (SEQ ID NO: 21)oligonucleotides as primers. The final 520 bp, 5 prime RACE product wassubcloned into the PCR II vector (Invitrogen, San Diego, Calif.) toyield pMON25433 and sequenced to obtain the remaining cDNA sequence (SEQID NO: 22). This sequence also displayed uninterrupted homology to thelysine oxidase genomic sequence. N10 (SEQ ID NO:16)CAT GTC GTC GAC GAG CAT GAG C N11 (SEQ ID NO:17)CAT CGA ACC CTT TGT CGA AGT CC N12 (SEQ ID NO:18)CAG CAA GCT TCT CTT TGT AAT ACC C N13 (SEQ ID NO:20)GTC GAA GTC CTC AGC CAG CTT CTC TTT GTA A N14 (SEQ ID NO:21)CAT GCT GGG GAT GTC AGG

[0110] To obtain a complete clone of the genomic DNA encoding the lysineoxidase, a lambda phage genomic DNA library (Frischauf et al., 1987)derived from F22844 was screened by hybridization with the lysineoxidase partial cDNA fragment. In brief, approximately 20,000 plaqueforming units from the library were plated, transferred to filters, andprobed with radiolabelled lysine oxidase cDNA fragment (SEQ ID NO: 12)which had been isolated from pMON23671. Briefly, filters were hybridizedwith a ³²P labeled probe (Feinberg and Vogelstein, 1983) with a specificactivity of approximately 2×108 DPM/μg in 5×SSC, 5×Denhardts, 0.1% SDS,50% formamide, and 500 μg/mL DNA at 42° C. for 18 hours, washed twice in2×SSC, 0.1% SDS for 15 minutes at 25° C. or room temperature, washedtwice in 0.1×SSC, 0.1% SDS for 20 minutes at 60° C. andautoradiographed. Positive or hybridizing plaques were then picked,re-plated , and re-probed until a purified isolate consisting of only ofhybridizing plaques is obtained. Five independent, hybridizing lambdaphage clones were obtained.

[0111] The DNA from the purified lambda phage was then prepared bystandard procedures and analyzed by both direct DNA sequencing andsouthern blot techniques. Sequencing of the lambda genomic clonesreveals essentially complete sequence homology to the partial lysineoxidase cDNA clone in pMON23671 and to one another, indicating that theclones are independent isolates of the same gene. Southern blot analysisof lambda phage genomic DNA digested with BamHI indicated that all ofthe clones carried a common cross hybridizing BglII fragment ofapproximately 5 kb and that genomic DNA of the F22844 has a similarband. The 5 kb BglII fragment from the lambda phage digest wassubsequently isolated and cloned into the BamHI site of pBSIIKS+(Stratagene, La Jolla, Calif.) to yield pMON23680 (FIG. 6). An internal1.0 kb Xbal fragment restriction mapped to the 3′ end of the genomic DNAwas deleted from pMON23680 to yield pMON23684 (FIG. 7). The completesequence of the genomic DNA encoding the lysine oxidase gene is given(SEQ ID NO: 15).

[0112] The complete sequence of the genomic clone of the F22844 lysineoxidase gene was determined and compared to the sequence of the lysineoxidase cDNA. Comparison of the genomic and cDNA sequence indicates thatthe lysine oxidase gene has no introns within its coding region. Morespecifically, the DNA sequences of the genomic DNA (in lambda clone 56-3and derived pMONs 23680 and 23684) were determined by automatedsequencing of both strands (Prism DyeDeoxy Cycle Sequencing-AppliedBiosystems, Foster City, Calif.) and confirmed with manual sequencing(Sanger dideoxy chain termination). Sequences of the cDNA fragments inpMONs 23671, 23681, 23683 and pMON25433 were obtained by automatedsequencing as well as by manual sequencing for pMON25433.

[0113] Analysis of the lysine oxidase genomic sequence shows a singleopen reading frame encoding a 617 amino acid residue ORF of approximatepredicted M_(r) 69,400. Since the native protein has an apparent M_(r)56,000 this ORF encodes a pre-protein that is post-translationallymodified to yield the mature protein. N-terminal sequence data and massspectroscopy data indicate that approximately 77 amino acid residues arecleaved from the N-terminus to yield the mature lysine oxidase proteinof approximate M_(r) 60,000.

[0114] A search of the SWISS PROT database with the entire 617 aminoacid residue lysine oxidase ORF (SEQ ID NO: 46; predicted M_(r)=69,400)encoded by the genomic sequence revealed homology of the Trichodermalysine oxidase to the Neurospora L-amino acid oxidase (LAO) precursorprotein (Niedermann and Lerch, 1990). The overall homology score was 24%identity, 50% similarity over 606 residues with the highest conservationin a region identified as a FAD binding site (8 of 9 contiguousresidues). Both lysine oxidase and LAO are apparently synthesized asproproteins since the first 129 amino acids of the non-secreted(intracellular) LAO proprotein are removed to yield the mature LAO(Niedermann and Lerch, 1990). This result suggests that the LAO ofNeurospora or other L-amino acid oxidases may be combined with theF22844 M_(r) 50,000 protein or other proteins to yield control of otherinsects.

[0115] The full length tedanalactam synthase cDNA was isolated in stagesusing PCR based protocols of mixed oligonucleotide primed amplificationof cDNA (MOPAC) (Lee et al., 1988) and rapid amplification of cDNA ends(R.A.C.E.) (Frohman et al., 1988). First strand cDNA was generated frompoly A+RNA isolated from 4 day old culture of F22844 and served astemplates for the MOPAC and RACE reactions. The first 5 prime (sense)gene specific amplification primer (GSP), primer N15 (SEQ ID NO: 23),was designed from the protein sequence P5 (SEQ ID NO: 24) obtained fromtryptic peptide fragment 11. A second nested 5 prime GSP, primer N16(SEQ ID NO: 25), was designed from protein sequence P6 (SEQ ID NO: 26)derived from tryptic fragment 9. The 3 prime (antisense) GSP, primer N17(SEQ ID NO: 27), was designed from the protein sequence P7 (SEQ ID NO:28) obtained from tryptic peptide fragment 16. A 623 bp partial cDNAfragment (SEQ ID NO: 29) was obtained from an RT-PCR reaction withprimers N16 (SEQ ID NO: 25) and N17 (SEQ ID NO: 27), and subcloned intopBluescript II KS+ (Stratagene, La Jolla, Calif.) at the SmaI site(blunt ligation) resulting in pMON25421 (FIG. 8). The partial cDNA wassequenced and the deduced protein sequence matched the protein sequenceobtained from tryptic fragments 9, 7, and 16 (SEQ ID NO: 26, SEQ ID NO:30,SEQ ID NO: 28). N15 (SEQ ID NO:23):GAR CAR AAY AAY TTY TTY AAY CAY GC P5  (SEQ ID NO:24):Val Val Val Leu Glu Gln Asn Asn Phe Phe Asn His Ala Gly SerSer Asn Asp Leu Ala N16 (SEQ ID NO:25): ATG TAY ACI GAR CAY TAY ATG P6 (SEQ ID NO:26): Thr Met Tyr Thr Glu Asp Tyr Met Ala Asp Leu Ala Lys N17(SEQ ID NO:27): GG IGC RAA YTG RAA CCA CAT P7  (SEQ ID NO:28):Gly Thr Ile Phe Pro Ser Met Trp Phe Gln Phe Ala Pro Asp Lys P8  (SEQ IDNO:30): Leu Gly Met Thr Tyr Gln Glu Met Ser Ala Lys

[0116] RACE was used to identify the remaining 5 prime and 3 prime cDNAsequence of the tedanalactam synthase gene. 5 prime RACE was performedaccording the Gibco-BRL kit using P6 (SEQ ID NO: 26) derived genespecific antisense primers N18 (SEQ ID NO: 31) and N19 (SEQ ID NO: 32).The 380 bp, 5 prime RACE product (SEQ ID NO: 33) was subcloned into thePCR II vector (Invitrogen, San Diego, Calif.) resulting in pMON25422(FIG. 9). To recover the 3 prime portion of the cDNA, 3 prime RACE wasperformed using a P6 (SEQ ID NO: 26) derived gene specific sense primerN20 (SEQ ID NO: 34) and the Universal Amplification Primer (Gibco-BRL,Gaithersburg, Md.) which generated a 1423 base pair fragment. The 1423bp fragment 3 prime race product (SEQ ID NO: 35) was subcloned into PCRII vector (Invitrogen, San Diego, Calif.) to yield pMON25423.

[0117] The full length 50 kb cDNA was generated by overlap PCR (Hortonet al., 1989) using F22844 first strand cDNA as template and primers:

[0118] 1) N21 (SEQ ID NO: 36), a 5 prime sense primer that introducesBglII and NcoI restriction sites at the start codon (ATG) (primermr50000-1)

[0119] 2) N22 (SEQ ID NO: 37) sense and N23 (SEQ ID NO: 38) antisense31-mers (primer mr50000-2 and mr50000-3) to remove the internal NcoIsite.

[0120] 3) N24 (SEQ ID NO: 39) an oligonucleotide that introduces a EcoRIand HindIII restriction sites 3 prime to the stop codon that is located1363 bp downstream of the start codon (ATG).

[0121] The engineered full length cDNA, 1385 bp PCR product (SEQ ID NO:40), was subcloned into PCR II vector (Invitrogen, San Diego, Calif.)resulting in pMON25424 (FIG. 10). The deduced translated proteinsequence is shown (SEQ ID: 41). N21 (SEQ ID NO:36):GGG AGA TCT CCA TGG CAG ACG AAA TCT N22 (SEQ ID NO:37):GGC TTT CCA GCA CTT CCT TGG GGC CCT CCA A N23 (SEQ ID NO:38):TTG GAG GGC CCC AAG GAA GTG CTG GAA AGC C N24 (SEQ ID NO:39):CCC AAG CTT GAA TTC ACT TTC TTC TAT TGC C

[0122] Genomic DNA was isolated from the fungal pellet of a 5 day oldliquid culture of F22844 (Fedoroff et al. 1983). Southern blot analysisindicated that the gene encoding tedanalactam synthase was a single copygene and mapped to an 8.0 kb BglII fragment. A F22844 genomic librarywas constructed from genomic DNA partially digested with MboI ligatedinto the BamHI site of the lambda EMBL3 vector (Frischauf et al., 1987).pMON 25421 cDNA insert was used to screen 48,000 plaque forming unitsfrom the primary library and nine positive overlapping clones wereidentified. The tedanalactam synthase gene was localized to a 9.0 kbSalI fragment. In three of the lambda clones, the gene mapped to one ofthe vector arms indicating that partial MboI digestions of F22844genomic DNA used in the construction of the library had createdtruncation of the 9.0 kb SalI fragment to a 6.0 kb, 4.4 kb and 2.5 kb.The 4.4 kb SalI fragment was subcloned into pBluescript II KS+(Stratagene, La Jolla, Calif.) resulting in pMON25425. The insert wassequenced and contained the complete 50 kb genomic clone with 5 introns(SEQ ID NO: 42).

Example 6

[0123] This example illustrates bioactivity of lysine oxidase andtedanalactam synthase derived from cloned genes against western cornrootworm.

[0124] The lysine oxidase and tedanalactam synthase genes can beisolated from novel sources or known sources. These genes may than beused to transform bacterial, yeast or plant cells, resulting in theproduction of lysine oxidase (or lysine oxidase proprotein) andtedanalactam synthase and permitting use of the methods of thisinvention. Examples of how this could be done for the lysine oxidase andtedanalactam synthase genes from fungal isolate F22844 are given below.

[0125] To introduce restriction endonuclease sites permitting expressionof the lysine oxidase structural gene in transformed microorganisms andplants, the cloned genomic DNA sequence of the F22844 lysine oxidasegene (SEQ ID NO: 15 in pMON23680 or 23684) was subjected to PCR mediatedsite-directed mutagenesis. Briefly, about 100 picograms of pMON23680 ina 100 μL reaction containing 1× Pfu buffer (Stratagene, La Jolla,Calif.), 100 μM dNTPs, 0.5 μM oligonucleotide primer N25 (SEQ ID NO:43), 0.5 μM oligonucleotide primer N26 (SEQ ID NO: 44), 2.5 units of Pfupolymerase (Stratagene, La Jolla, Calif.) was overlaid with mineral oiland subjected to thermal cycling in a Perkin Elmer Model 480 DNA ThermoCycler. The thermal cycling profile was 1 cycle at 94° C. for 1.5minutes, 50° C. for 1 minute, 74° C. for 3 minutes followed by 25 cyclesof 94° C. for 1 minute, 50° C. for 1 minute, 74° C. for 2 minutes andterminated with a final cycle of 94° C. for 1 minute, 50° C. for 1minute, 74° C. for 15 minutes. The PCR reaction product of approximately1,900 bp was electrophoresed on an agarose gel, purified (Qiagen,Chatsworth, Calif.), and digested with restriction endonucleases NcoIand EcoRI. The sequence of the resultant PCR product is given (SEQ IDNO: 45). Note that the amino acid sequence of the lysine oxidase M_(r)69,400 proprotein (SEQ ID NO: 46) encoded by the mutagenized DNAsequence is identical to the deduced translation product ofunmutagenized lysine oxidase genomic sequence (SEQ ID NO: 15;translation start at base number 663, translational stop at base number2513). N25 (SEQ ID NO:43): TTGCAAACCATGGACAATGTTGACTTTGCTGAATC N25 (SEQID NO:44): GCCGTAGTACCGAATTCTTATTAAATCTTCACC

[0126] Convenient restriction sites for placing the tedanalactamsynthase gene into plant expression vectors were also introduced by PCRsite-directed mutagenesis as described previously.

[0127] To obtain functional lysine oxidase protein from the cloned gene,the lysine oxidase gene was engineered for expression in a heterologousyeast system. The yeast expression construct, pMON25030 (FIG. 11), wasmade by cloning a PCR generated fragment encoding the lysine oxidaseM_(r) 69,400 proprotein (SEQ ID NO: 46) into a pYES2 yeast expressionplasmid (Invitrogen, San Diego, Calif.). The PCR fragment (approximately1.9 kb) was generated by using two primers, N26 (SEQ ID NO: 47) and N27(SEQ ID NO: 48) and PCR amplifying a segment of DNA from pMON23680 (SEQID NO: 15). Each primer carried a unique restriction site which provideddirectional cloning of the fragment into the pYES2 vector under GALlpromoter control. Primer N26 (SEQ ID NO: 47) carried a BglII site whileN27 (SEQ ID NO: 48) had an XbaI site. The resulting PCR fragment wasdigested with BglII and XbaI and cloned into BamHI and XbaI sites of thepYES2 vector. Note that the amino acid sequence of the lysine oxidaseM_(r) 69,400 proprotein (SEQ ID NO: 46) encoded by this DNA fragment isnot affected by introduction of these restriction sites. N26 (SEQ IDNO:47): CCCAGATCTATATTTGCAAACATGGACAATG N27 (SEQ ID NO:48):GGGTCTAGACTAACAAACATCACACTTTCTATG

[0128] Yeast (Saccharomyces cerevisiae) transformed with pMON25030 weregrown in the presence of galactose and shown to produce enzymaticallyactive lysine oxidase. Yeast transformed with pMON25030 had solublelysine oxidase activity in both disrupted cell pellets and cell freeculture media. Culture media incubated for five days of galactoseinduction was found to hold the majority of lysine oxidase activity atapproximately 1 ng equivalent of lysine oxidase activity per 1 μl ofmedia (activity units are standardized to the activity of known amountsof F22844 lysine oxidase). Lysine oxidase from the yeast culture mediawas subsequently purified and shown to have bioactivity against cornrootworm when combined with tedanalactam synthase. Surviving larvaeexhibit 51% stunting with the combination sample of recombinant lysineoxidase purified from yeast and the F22844 culture filtrate purifiedtedanalactam synthase (Table 11). No significant stunting was seen witheither protein individually. The yeast purified lysine oxidase had aspecific activity of 25 U/mg protein versus a specific activity of 37U/mg protein when the enzyme is purified from F22844 TABLE 11 TreatmentConcentration # of Mean larval wt. (16 wells/sample) (ppm) survivors(mg) (±SEM) Acetate buffer — 61 0.94 ± 0.08 Acetate buffer — 45 0.84 ±0.07 Lysine oxidase 14 38 0.76 ± 0.06 M_(r) 50,000 10 43 0.80 ± 0.07Lysine oxidase + 14 + 10 36 0.44 ± 0.05 M_(r) 50,000

[0129] An NcoI-HindIII fragment containing the full length tedanalactamsynthase cDNA gene (SEQ ID NO: 40) from pMON25424 was inserted intopMON6235, an E. coli expression vector containing an arabinose induciblepromoter and GIG leader sequence to produce pMON25428 (FIG. 12). Westernblot analysis showed that the E. coli expressed protein was crossreactive to polyclonal antibodies raised against the F22844 purifiedtedanalactam synthase. The relative migration of the expressed proteinon SDS-PAGE was identical to the F22844 purified tedanalactam synthase.The recombinant tedanalactam synthase was purified in a 5 step procedurefrom E. coli expressing the tedanalactam synthase gene. Lysine oxidasewas purified from a commercial preparation of T. viride lysine oxidase.Both the lysine oxidase and tedanalactam synthase were >95% pure bySDS-PAGE. In bioassays against SCRW neonate larvae, >80% stunting ofsurvivors was seen with the combination sample of T. viride lysineoxidase and the E. coli produced F22844 tedanalactam synthase (Table12). The number of survivors was also dramatically reduced. Nosignificant mortality or stunting was seen with either proteinindividually. This demonstrates that the heterologously expressed orrecombinant tedanalactam synthase is insecticidally active against cornrootworm when assayed in the presence of lysine oxidase. TABLE 12Treatment (16 Concentration Mean larval wells/sample) (ppm) # ofsurvivors wt. (mg) (+SEM) Tris/acetate buffer — 51 0.68 ± 0.04Tris/acetate buffer — 52 0.57 ± 0.04 Lysine oxidase 10 46 0.48 ± 0.04M_(r) 50,000 18 80 0.52 ± 0.03 Lysine oxidase + 10 + 18 23 0.11 ± 0.01M_(r) 50,000

[0130] Lysine oxidase isolated from yeast transformed with pMON25030 andtedanalactam synthase isolated from E. coli transformed with pMON25428were tested in bioassays for activity against western corn rootworm(Diabrotica) when combined respectively with tedanalactam synthase orlysine oxidase from F22844. Concentration response curves were run todetermine the efficacy of these purified proteins on neonate WCRW larvae(Table 13). Excellent bioactivity was observed with LC₅₀'s below 2 ppmfor each protein. TABLE 13 Sample Conc. (ppm) % Mortality % StuntingF50/FLO¹ 20 + 20 100 — 10 + 10 100 — 2 + 2 63 80 F50/YLO² 20 + 20 100 —10 + 10 100 — 2 + 2 90 87 E50/FLO³ 10 + 10 84 80

Example 7

[0131] This example illustrates the production of antibodies useful fordetection and quantitation of lysine oxidase and tedanalactam synthase

[0132] To detect and quantitate tedanalactam synthase, polyclonalantibody #208 was developed by immunizing a rabbit with partiallypurified 50K protein derived from the E. coli expression vectorpMON25428. A quantitative Enzyme Linked Immunoassay (ELISA) wherepolyclonal antibody #208 is both the coating and a Horse RadishPeroxidase (HRP) conjugated antibody was devised. The linear range ofthe tedanalactam synthase ELISA is between approximately 2.5 to 40 ng .Antibody #208 is also used for 50K Western blot analysis using theEnhanced Chemi-Luminescence (ECL) method of Amersham (cat #RPN 2106).The Western blot procedure utilizes antibody #208 as the primaryantibody and an Amersham goat anti-rabbit HRP conjugated secondaryantibody (cat #NA 934). Incubation in the ECL reagents allowsvisualization of the antigens by autoradiography.

[0133] To detect and quantitate lysine oxidase, polyclonal antibody #450was developed by immunizing a rabbit with partially purified lysineoxidase protein derived from the S. cerevisiae expression vectorpMON25030. A quantitative Enzyme Linked Immunoassay (ELISA) for lysineoxidase where polyclonal antibody #450 is both the coating andHRP-conjugated antibody was devised. The linear range of the lysineoxidase ELISA is between approximately 0.25 to 4.0 ng.

[0134] For analysis of the lysine oxidase by Western blot analysis,polyclonal antibody #2262 sera #3 was developed by immunizing a rabbitwith a KLH conjugated 25-mer peptide CSVGEKLQQAFGYYKEKLAEDFDKG where allbut the N-terminal cysteine residue is derived from the lysine oxidasepeptide sequence. This antibody will detect both the lysine oxidaseproenzyme of approximate M_(r) 69,000 as well as the enzymaticallyactive and mature form of approximate M_(r) 69,000. Antibody #2262 sera#3 is used for LO Western blot analysis using the EnhancedChemi-Luminescence (ECL) method of Amersham (cat #RPN 2106). The Westernblot procedure utilizes antibody #2262 sera #3 as the primary antibodyand an Amersham goat anti-rabbit HRP conjugate as the secondary antibody(cat #NA 934). Incubation in the ECL reagents allows visualization ofthe antigens by autoradiography.

Example 8

[0135] This example illustrates control of insects by expression oflysine and M_(r) 50,000 proteins in plant colonizing bacteria.

[0136] To control insects, it may be desirable to express lysine oxidaseand tedanalactam synthase in plant colonizing bacteria, and then applythis bacteria to the plant. As the insect feeds on the plant, it ingestsa toxic dose of lysine oxidase and tedanalactam synthase produced by theplant colonizers. Plant colonizers can be either those that inhabit theplant surface, such as Pseudomonas or Agrobacterium species, orendophytes that inhabit the plant vasculature such as Clavibacterspecies. For surface colonizers, the lysine oxidase and tedanalactamsynthase genes may be inserted into a broad host range vector capable ofreplicating in these Gram-negative hosts. Examples of these such vectorsare pKT231 of the IncQ incompatibility group (Bagdasarian et al., 1981)or pVK100 of the IncP group (Knauf and Nester, 1982). For endophytes thetedanalactam synthase and lysine oxidase genes can be inserted into thechromosome by homologous recombination or by incorporation of the geneonto an appropriate transposon capable of chromosomal insertion in theseendophytic bacteria.

Example 9

[0137] This example illustrates control of coleopteran insects byexpression of lysine oxidase and tedanalactam synthase in transgenicmonocotlyedenous plants.

[0138] To place the lysine oxidase gene in a vector suitable forexpression in monocotyledonous plants (i.e. under control of theenhanced Cauliflower Mosaic Virus 35S promoter and linker to the hsp70intron followed by a nopaline synthase polyadenylation site as in Brownand Santino, U.S. Pat. No. 5,424,412; 1995), the vector pMON19469 (FIG.13) was digested with NcoI and EcoRI. The larger vector band ofapproximately 4.6 kb was electrophoresed, purified, and ligated with T4DNA ligase to the Ncol-EcoRI fragment of approximately 1.9 kb containingthe lysine oxidase gene (SEQ ID NO: 45). The ligation mix wastransformed into E. coli strain XL-1 Blue (Stratagene, La Jolla,Calif.). Carbenicillin resistant colonies were recovered and plasmid DNArecovered by DNA miniprep procedures. This DNA was subjected torestriction endonuclease analysis with enzymes such as NcoI and EcoRI(together), NotI, and PstI to identify plasmid pMON25040, which containsthe lysine oxidase coding sequence fused to the hsp70 intron undercontrol of the enhanced CaMV35S promoter (FIG. 14). Expression offunctional lysine oxidase by pMON25040 in corn protoplasts was confirmedby electroporation of pMON25040 DNA into protoplasts followed byenzymatic assays of the plant protoplasts for lysine oxidase activity.

[0139] To place the lysine oxidase gene in a vector suitable forrecovery of stably transformed and insect resistant plants, the 3.6 kbNotl restriction fragment from pMON25040 containing the lysine oxidasecoding sequence fused to the hsp70 intron under control of the enhancedCaMV35S promoter was isolated by gel electrophoresis and purification.This fragment was ligated into NotI digested, alkaline phosphatasetreated pMON15786, a plant transient expression vector containing theneomycin phosphotransferase coding sequence fused to the hsp70 intronunder control of the enhanced CaMV35S promoter (FIG. 15). Kanamycinresistant colonies were obtained by transformation of this ligation mixinto E. coli XL-1 Blue (Stratagene, La Jolla, Calif.) and coloniescontaining pMON25041 (FIG. 16) identified by restriction endonucleasedigestion of plasmid miniprep DNAs. Restriction enzymes such as NotI,EcoRV, HindIII, NcoI, EcoRI, and BglII can be used to identify theappropriate clones containing the NotI fragment of pMON25040 in the NotIsite of pMON15786 (i.e. pMON25041) in the orientation such that bothgenes are in tandem (i.e. the 3′ end of the lysine oxidase expressioncassette is linked to the 5′ end of the nptII expression cassette). Thisvector can be introduced into the genomic DNA of corn embryos byparticle gun bombardment followed by paromomycin selection to obtaincorn plants expressing the lysine oxidase gene essentially as describedin Brown and Santino U.S. Pat. No. 5,424,412. These plants can then be“crossed” by pollen transfer to plants containing the tedanalactamsynthase gene (construction described below, from pMON30411) to obtainplants that are resistant to insect infestation, particularly cornrootworm (Diabrotica spp.). Alternatively, pMON30411 and 25040 could beco-bombarded to obtain plants that are resistant to insect infestation,particularly corn rootworm (Diabrotica spp.).

[0140] To place the tedanalactam synthase gene in a vector suitable forexpression in monocotyledonous plants (i.e. under control of theenhanced Cauliflower Mosaic Virus 35S promoter and linked to the hsp70intron followed by a nopaline synthase polyadenylation site), the vectorpMON19469 was digested with NcoI and EcoRI. The larger vector band ofapproximately 4.6 kb was electrophoresed, purified, and ligated with T4DNA ligase to the NcoI-EcoRI fragment of approximately 1.4 kb containingthe tedanalactam synthase gene (SEQ ID NO: 40) obtained from the E. coliexpression vector pMON25428. The ligation mix was transformed into E.coli strain XL-1 Blue (Stratagene, La Jolla, Calif.), carbenicillinresistant colonies recovered and plasmid DNA recovered by DNA miniprepprocedures. This DNA was subjected to restriction endonuclease analysiswith NcoI and EcoRI, NotI, and SacI to identify clones containingpMON30410, containing the tedanalactam synthase coding sequence fused tothe hsp70 intron under control of the enhanced CaMV35S promoter (FIG.18). Expression of the tedanalactam synthase gene by pMON30410 in cornprotoplasts was confirmed by electroporation of pMON30410 DNA intoprotoplasts followed by Western blot analysis of the plant protoplastextracts for tedanalactam synthase cross reacting species of the same Mras native tedanalactam synthase.

[0141] To place the tedanalactam synthase gene in a vector suitable forrecovery of stably transformed and insect resistant monocot plants, the3.1 kb NotI restriction fragment from pMON30410 containing thetedanalactam synthase coding sequence fused to the hsp70 intron undercontrol of the enhanced CaMV35S promoter was isolated by gelelectrophoresis and purification. This fragment was ligated withpMON15786 treated with NotI and calf intestinal alkaline phosphatase(pMON15786 contains the neomycin phosphotransferase coding sequencefused to the hsp70 intron under control of the enhanced CaMV35Spromoter). Kanamycin resistant colonies were obtained by transformationof this ligation mix into E. coli XL-1 Blue (Stratagene, La Jolla,Calif.) and colonies containing pMON30411 (FIG. 17) identified byrestriction endonuclease digestion of plasmid miniprep DNAs. Restrictionenzymes such as NotI, EcoRV, HindIII, NcoI, EcoRI, and BglII may be usedto identify the appropriate clones containing the NotI fragment ofpMON30411 in the NotI site of pMON15786 (i.e. pMON30411) in theorientation such that both genes are in tandem (i.e. the 3′ end of thetedanalactam synthase expression cassette is linked to the 5′ end of thenptII expression cassette). This vector can be introduced into thegenomic DNA of corn embryos by particle gun bombardment followed byparomomycin selection to obtain corn plants expressing the tedanalactamsynthase gene. These plants can then be “crossed” by pollen transfer toplants containing the lysine oxidase gene (pMON25041; see descriptionbelow) to obtain plants that are resistant to insect infestation,particularly corn rootworm (Diabrotica spp.). Alternatively, pMON30411and 25040 could be co-bombarded to obtain corn plants that are resistantto insect infestation, particularly corn rootworm (Diabrotica spp.).

[0142] To place both the tedanalactam synthase and lysine oxidase genesin a vector suitable for recovery of stably transformed and insectresistant plants, the 3.6 kb NotI restriction fragment from pMON25040containing the lysine oxidase coding sequence fused to the hsp70 intronunder control of the enhanced CaMV35S promoter was isolated by gelelectrophoresis and purification. This fragment was ligated withpMON30411 that was partially digested with NotI to obtain pMON30411plasmid DNA cut only once with NotI and treated with calf intestinalalkaline phosphatase (pMON30411 contains the neomycin phosphotransferasecoding sequence fused to the hsp70 intron under control of the enhancedCaMV35S promoter and the tedanalactam synthase coding sequence fused tothe hsp70 intron under control of the enhanced CaMV35S promoter).Partial digestion and isolation of pMON30411 plasmid with only one NotIsite cut was accomplished by digesting 5 μg of DNA in 1×HSB buffer(Boehringer-Mannheim, Indianapolis, Ind.), 10 μg/mL bovine serumalbumin, 10 μg/mL ethidium bromide and 0.4 units of NotI restrictionendonuclease followed by gel electrophoresis and purification of thelinearized plasmid DNA. Kanamycin resistant colonies were obtained bytransformation of this ligation mix into E. coil XL-1 Blue (Stratagene,La Jolla, Calif.). Colonies containing pMON30417 (FIG. 19) wereidentified by restriction endonuclease digestion of plasmid miniprepDNAs. Restriction enzymes such as NotI, Sacl, HindIII/BamHI, NcoI/EcoRI,EcoRV and BglII may be used to identify the appropriate clonescontaining the NotI fragment of pMON25040 in the NotI site of pMON30411upstream of the tedanalactam synthase gene (i.e. pMON30417) in theorientation such that all genes are in tandem (i.e. the 3′ end of thelysine oxidase cassette is linked to the 5′ end of the tedanalactamsynthase expression cassette and the 3′ end of the M_(r) 50,000expression cassette is linked to the 5′ end of the nptII expressioncassette). Expression of both the lysine oxidase and tedanalactamsynthase in genes in corn leaf protoplasts electroporated with pMON30417was observed. This vector can be introduced into the genomic DNA of cornembryos by particle gun bombardment followed by paromomycin selection toobtain corn plants expressing both the lysine oxidase and tedanalactamsynthase genes essentially as described in Brown and Santino U.S. Pat.No. 5,424,412. Transgenic corn plants expressing the lysine oxidase andtedanalactam synthase genes can be identified by an ELISA assay specificfor the two proteins or via an enzymatic assay for lysine oxidaseactivity. These plants may be resistant to insect infestation,particularly corn rootworm (Diabrotica spp.).

[0143] Both the lysine oxidase gene in pMON25040 and the tedanalactamsynthase gene in pMON30410 were shown to express the respective F22844genes in corn leaf protoplasts. Corn leaf protoplasts wereelectroporated with pMONs 25040 and 30410 as well as a pMON19649 controland incubated for about 24 hours. Total protein was extracted andassayed for the presence of enzymatic lysine oxidase activity (Table 14)or tedanalactam synthase cross reacting material by Western blotanalysis. Both genes are expressed in corn cells. TABLE 14 ng LOactivity/mg total protein¹ Vector cell pellet² culture media (conc)³pMON25040 148 ± 12 411 ± 3 pMON19649 0 0

[0144] Corn plants transformed with the monocot transformation vectorcontaining both the lysine oxidase and tedanalactam synthase expressioncassettes (i.e. pMON30417) were obtained as described above. Atransgenic event that expressed both lysine oxidase and tedanalactamsynthase was identified and outcrossed to yield progeny that expressboth the lysine oxidase and tedanalactam synthase proteins atapproximately 2.5 and 4.5 PPM, respectively (leaf expression levels asdetermined by an ELISA). Progeny plants of the pMON30417 event andgenotypically identical controls that do not express the lysine oxidaseor tedanalactam synthase proteins were infested with western cornrootworm eggs and scored for root damage by the Iowa Rating system(Hills and Peters, 1971) after 3 weeks of feeding. These results aresummarized in Table 15. TABLE 15 pMON30417 mediated control of westerncorn rootworm in transgenic corn Treatment N¹ Mean RDR² (SE)³ Range⁴pMON30417 10 3.0 (0)   3 Control 5 4.6 (0.24) 4-5

[0145] In addition to the quantitative Root Damage Rating datademonstrating protection of corn plant roots expressing the lysineoxidase and tedanalactam synthase proteins (pMON30417), a number ofqualitative observations also indicate that these genes confer cornrootworm control. First, the above ground nodal roots were intact in thepMON30417 plants but were destroyed in the control plants. Second, thecontrol plants had copious numbers of larvae within the stalk at thetime of examination while the pMON30417 plants had none.

[0146] A bare root assay was performed on segregating ‘Laffite’ plantsto measure plant insecticidal activity and larval growth. Survival onthe control was 39% as compared to 4% on F22844. Those surviving onF22844 were in very poor condition (Table 16). Because of the poorcondition of the F22844 larvae, no larval weights were recovered. TABLE16 Percent survival and mean larval weight of WCR in a bare root assay.Treatment N Survival (%) Larval weight (mg) Control 15 38.7 0.24 F228449 4.4 —

[0147] The western corn rootworm whole plant bioassay was repeated with‘Laffite’ in ten inch pots. The larger pot would allow for near normalplant development, thus allowing for both the opportunity to studyinsect control and the phytotoxic effects. Positive plants (2-4 ppm)were approximately 30-40 percent stunted at the end of the assay periodfor the H99 and B73 pedigrees, respectively. The root system on thepositive plants appeared normal for that size of a plant. The insectcontrol on the positive plants appeared normal for that size of a plant.

[0148] The average root damage ratings of the F22844 plants weresignificantly lower than the negative plants, 2.6 and 2.7 versus 5.2 and5.9 for the H99 and B73 pedigrees, respectively. The negative segregantsshowed severe rootworm damage with 2-3 entire nodes of the roots pruned(Table 17). Note that constitutive expression of enzymatically active LOcan result in plant height reductions (Table 17). Combinations of rootspecific promoters, LO69 proenzyme variants resistant to plant proteaseactivation, and/or organellar or extracellular targeting of LO69 or LO69proenzyme variants are anticipated to relieve this effect of LOexpression in plants. TABLE 17 Mean root ratings (RDR) for F22844, Event‘Laffite’, for both a B73 and H99 S1 cross from the R0 in a ten inch potWCR bioassay Plant Height Treatment Pedigree N RDR Mean RDR Range Mean(inch) F22844 H99 16 2.6 1-3 28.0 B73 10 2.7 1-3 30.6 Control H99 10 5.23-5 39.3 B73  8 5.9 5-6 49.6

[0149] To reduce plant height reductions caused by lysine oxidaseexpression and to target expression of the insecticidal proteins to theroots attacked by Diabrotica sp, promoters that limit expression oflysine oxidase to roots are valuable. In this example, the 4 as-1 rootenhanced promoter (Lam et al., U.S. Pat. No. 5,023,179; designated pAS4in plasmid maps) was fused to a transcription unit containing the maizehsp70 intron, the lysine oxidase gene, and the nos polyadenylation sitein pMON25058 (FIG. 20). The 4 as-1 promoter was also fused to atranscription unit containing the maize hsp70 intron, the tedanalactamsynthase gene, and the nos polyadenylation site in pMON25060. Both thelysine oxidase and tedanalactam synthase transcription units is weresubsequently combined in a vector containing a neomycinphosphotransferase coding sequence fused to the hsp70 intron undercontrol of the enhanced CaMV35S promoter to yield pMON25061 (FIG. 21).Transgenic corn plants expressing both lysine oxidase and tedanalactamsynthase genes were recovered as previously described.

[0150] One illustrative pMON25061 event, R44482, was further analyzedand shown to express lysine oxidase and tedanalactam synthase atrelatively high levels in root tissue and at relatively low levels inleaf tissue (2.9 ppm lysine oxidase in root versus 0.9 ppm in leaf; 8.4ppm tedanalactam synthase in root versus 1.9 ppm in leaf). Event 44482was also displayed significant levels of CRW resistance in both growthchamber and field tests (Table 18). Although plant height reductionsassociated with the pMON25061 transgene in R44482 were not eliminated,the level of stuntung is clearly less than that observed when Lysineoxidase is expressed from a constitutive CaMV e35S promoter (Tables 17and 18). TABLE 18 Corn Rootworm Damage Rating and Height of CRW infestedand non-infested pMON25061 plants and wild type controls CRW RDR¹ CRWRDR¹ CRW RDR¹ (Height) [Growth (Height) [Field (Height) [Field PlantsChamber] Test Site #1] Test Site #2] 25061#44482  3.2 RDR 5.8 RDR 5.6RDR (infested) (51.5 in) (47 in) (37 in) wild type 10.4 RDR 9.4 RDR 11.9RDR (infested) (50.6 in) (60 in) (51 in) 25061#44482 ND ND ND(uninfested) (79 in) (84 in) wild type ND ND ND (uninfested) (84 in) (86in)

Example 10

[0151] This example illustrates construction of lysine oxidase andtedanalactam synthase dicot plant expression vectors, production ofdicot plants expressing these genes, and use of these plants to controlinsect pests.

[0152] To control coleopteran insect pests such as the Colorado potatobeetle, Leptinotarsa decemlineata (Say), in dicotyledonous plants suchas potato, Solanum tuberosum, or to control boll weevil, Anthonomusgrandis, in cotton, Gossypium hirsutum, it would be useful to engineerthe lysine oxidase and tedanalactam synthase genes for expression indicotyledonous plants.

[0153] To express tedanalactam synthase in dicots, the HindIII/Ncolfragment containing a duplicated or enhanced version of the FMV promoter(Rogers, 1995) and petunia hsp70 5 prime untranslated leader (Winter etal., 1988) from pMON18411 was ligated into the NcoI/HindIII sites ofpMON30410 located 5 prime to the tedanalactam synthase coding region(SEQ ID NO: 40) to yield pMON25043, containing a tedanalactam synthasevariant under the control of an enhanced FMV promoter/petunia hsp70untranslated leader with a NOS polyadenylation signal (FIG. 21). Thecomposite eFMV/hsp70/tedanalactam synthase/nos gene cassette wasisolated as a HindIII/NotI fragment from pMON25043 and ligated into theHindIII and NotI sites in pMON10098, a double border planttransformation vector(FIG. 22), to form pMON25046 (FIG. 23). ThepMON25046 plasmid is an Agrobacterium-based plant transformation vectorthat places the composite eFMV/hsp70/tedanalactam synthase/nos gene andenhanced CaMV35S/neomycin phosphotransferase/nos kanamycin selectablemarker gene between the right and left T-DNA border fragments. Thisvector can be mobilized into Agrobacterium (Ditta et al., 1980) and usedto obtain transgenic dicotyledonous plants as described (Horsch et al.,1985).

[0154] To express lysine oxidase in dicots, the NcoI/SmaI fragment ofthe lysine oxidase gene from pMON25040 (SEQ ID NO: 45) was ligated intothe NcoI and SmaI sites located between the enhanced FMVpromoter/petunia hsp70 5 prime untranslated leader (utl or UTR) and theNOS polyadenylation signal to yield pMON25042, containing a lysineoxidase variant under the control of the enhanced FMV promoter/petuniahsp70 leader with a NOS polyadenylation signal (FIG. 24). The compositeeFMV/hsp70 utl/lysine oxidase/nos gene from pMON25042 was isolated frompMON25042 on a single NotI fragment and subsequently ligated into theNotI site of pMON25048, a derivative of pMON10098, containing a singleNotI site and kanamycin selectable marker gene located between the rightand left T-DNA border sequences, to obtain pMON25050 (FIG. 25).pMON25050 can be mobilized into Agrobacterium (Ditta et al., 1980) andused to obtain transgenic dicotyledonous plants as described previously(Horsch et al., 1985).

[0155] To place both the tedanalactam synthase and lysine oxidase genesin a vector suitable for recovery of stably transformed and insectresistant dicotyledonous plants, the NotI fragment containing the lysineoxidase expression cassette from pMON25042 was ligated into a singleNotI site in pMON25046 introduced by single hit partial NotI digestionto obtain pMON25049 (FIG. 2). The pMON25049 plasmid is an Agrobacteriummediated transformation vector containing a kanamycin selectable markergene, the tedanalactam synthase expression cassette, and the lysineoxidase expression cassette between the right and left T-DNA bordersequences. This vector can be used to obtain dicotyledonous plantsexpressing both the tedanalactam synthase and lysine oxidase genes viathe previously described Agrobacterium-mediated plant transformationtechniques.

[0156] Arabidopsis plants were transformed with pMON25046 and pMON25050via the Agrobacterium mediated meristem infiltration technique (Bechtoldet al., 1993) and screened for expression of the tedanalactam synthaseand lysine oxidase genes, respectively. Identification of tedanalactamsynthase expressors was via an ELISA assay specific for the tedanalactamsynthase protein. Lysine oxidase expressors were identified via directenzymatic assays. Pollen was transferred from lysine oxidase expressingpMON25050 lines to tedanalactam synthase expressing pMON25046 lines toobtain F1 progeny that express both the tedanalactam synthase and lysineoxidase genes. These F1 progeny plants that expressed both the lysineoxidase and tedanalactam synthase genes and appropriate controls wereexposed to southern corn rootworm larvae to determine if these geneswould cause larval stunting (as assayed by reductions in larval weightpost-feeding) and reduced feeding (as assayed by a Leaf Damage Rating).The results displayed in Table 19 demonstrate that these genes causesignificant larval stunting and yield reduced leaf damage when expressedin transgenic Arabidopsis plants. TABLE 19 Activity of F22844 expressingArabidopsis on SCRW as shown by mean larval weight (mg) and Leaf DamageRating (LDR). N is the number of larvae weighed. Treatment N Mean Larvalwt. (mg) (±SEM) Mean LDR LO + M_(r) 50,000 25 0.23 (±0.02) 0.84untransformed 49 0.44 (±0.02) 2.79 LO only 25 0.47 (±0.03) 2.11 M_(r)50,000 only 37 0.39 (±0.02) 2.00

[0157] It may also be advantageous to localize the lysine oxidase ortedanalactam synthase to the plastids. Proteins can be directed to thechloroplast by including at their N-termini a chloroplast transitpeptide (CTP). One CTP that has worked to localize heterologous proteinsto the chloroplast of dicotyledonous plants is from the RUBISCO smallsubunit gene of Arabidopsis, denoted ats1A. A variant of this transitpeptide that encodes the transit peptide, 23 amino acids of matureRUBISCO sequence, plus a reiteration of the transit peptide cleavagesite has been constructed for the successful chloroplast localization ofthe B.t.k protein (Fischhoff and Perlak, 1990). It is anticipated thatthis same fragment of the ats1a gene, when fused in frame to theamino-terminal coding region of the lysine oxidase or tedanalactamsynthase genes, will similarly result in chloroplast localization ofthese proteins in dicotyledonous plants. Note that this chloroplasttargeted expression cassette is subsequently cloned into a binaryAgrobacterium transformation vector, mobilized into disarmedAgrobacterium hosts and used to transform dicots.

[0158] To localize lysine oxidase in corn plastids, A fragment of themaize RUBISCO small subunit gene denoted mSSU CTP or zmSl containing aduplicated cleavage site (Russell et al., 1993) was used to localizeeither the lysine oxidase or tedanalactam synthase in chloroplasts ofcorn plants. For example, an Xbal/Ncol fragment (SEQ ID NO: 49) encodingthe mSSU CTP derived from pMON22089 was ligated into the Xbal and NcoIsites of pMON30405 (lysine oxidase; SEQ ID NO: 50) or pMON30410 (M_(r)50,000 protein) that are located between the hsp70 intron and codingregions of these genes to obtain in-frame fusions of the mSSU CTP to theN-termini of these proteins. The pMON30405 lysine oxidase sequence (SEQID NO: 50) was obtained by PCR mutagenesis using pMON23684 (SEQ ID NO:15) as template with oligonucleotides N28 (SEQ ID NO: 51) and N29 (SEQID NO: 52) as primers. The mSSU CTP fusions to lysine oxidase or M_(r)50,000 protein under control of the enhanced CaMV35S promoter and thehsp70 intron are cloned as NotI fragments into pMON15786, a monocottransformation vector described above. Transgenic corn plants expressinglysine oxidase were obtained at a lower frequency when the plastidtargeting signal was employed, indicating that plastid targeting is nota preferred method of expressing lysine oxidase in transgenic corn.

Example 11

[0159] This example illustrates mitochondrial targeting of thetedanalactam synthase or lysine oxidase proteins in transgenic plants.

[0160] It may also be advantageous to target the tedanalactam synthaseor lysine oxidase proteins to the mitochondria. This may be accomplishedby fusing a suitable mitochondrial targeting peptide (MTP) to theamino-terminus of either the tedanalactam synthase or lysine oxidaseproteins. One example of an MTP that has been demonstrated to targetheterologous proteins to mitochondria of dicots is from the beta subunitof the mitochondrial ATP synthase (Boutry et al., 1987). We infer thatthis same sequence will direct mitochondrial import of either thetedanalactam synthase or lysine oxidase proteins in transgenic dicotplants when fused in frame to the amino-termini of these proteins.Appropriate promoter and termination sequences, Agrobacterium vectors,and transformation procedures needed to obtain transgenic dicotyledonousplants expressing the MTP fusion genes were described previously.

[0161] It may similarly be desirable to target the tedanalactam synthaseor lysine oxidase proteins to the mitochondria of monocotyledonousplants by making fusions of monocotyledonous plant derived MTPs to theseproteins. One example of a sequence that may direct mitochondrial importin monocots is one that is substantially homologous to the maizemitochondrial ATP synthase beta subunit (Winning et al., 1990). Anotherexample would be the MTP from the maize superoxide dismutase isozyme 3(Sod3) (White and Scandalios, 1989).

[0162] For example, the XbaI/NcoI fragment containing the portion of themaize ATP synthase beta subunit MTP sufficient to direct mitochondrialimport (SEQ ID NO: 53) from pMON30447 was ligated into the XbaI and NcoIsites of pMON25058 (lysine oxidase) that are located between the hsp70intron and lysine oxidase coding region to obtain in-frame fusions ofthe maize ATP synthase beta subunit MTP (zmBATP) to the N-termini oflysine oxidase, producing plasmid pMON33700 (FIG. 26). The pMON33700NotI cassette containing the zmBATP-lysine oxidase expression cassettewas engineered into pMON33701 (FIG. 27) to obtain pMON33702, a planttransformation vector containing the zmBATP-Lysine oxidase expressioncassette as well as previously described neomycin phosphotransferase andtedanalactam synthase expression cassettes (FIG. 28).

[0163] Transgenic corn expressing both lysine oxidase and tedanalactamsynthase in roots were obtained with pMON33702. Phenotypic analysis ofthese lines indicated that use of the zmBATP targeting signal did notalleviate stunting associated with high level lysine oxidase expressionin leaves. However, root specific or enhanced expression of themitochondrial targeted lysine oxidase expression may alleviate stunting.It is also possible that a strategy similar to that described abovecould be used in combination with plant protease insensitive variants oflysine oxidase proenzyme to obtain transgenic maize that are notstunted.

Example 12

[0164] This example illustrates apoplastic, vacuolar, endoplasticreticulum, and peroxisomal targeting of lysine oxidase proenzyme orenzyme.

[0165] To target the lysine oxidase proprotein to the extracellular orapoplastic space, a secretory signal peptide sequence derived fromplants can be fused in frame to the amino terminus of the lysine oxidaseproprotein gene. One example of such a sequence is the signal peptidederived from a barley cysteine endoproteinase gene (Koehler and Ho,1990). Another example is the tobacco PRIb signal peptide (Cornelissenet al., 1986).

[0166] It is also recognized that both the lysine oxidase andtedanalactam synthase proteins contain N-linked glycosylation sites thatare not used in the native proteins derived from Trichoderma. To avoidinactivation caused by N-glycosylation of secreted lysine oxidase ortedanalactam synthase, the glycosylation sites of these proteins couldbe eliminated by site directed mutagenesis. More specifically, all or asubset of the amino acid sequences N(×)S/T could be converted to N(×)Aby replacing the native Ser or Thr codon for an Ala codon. For thelysine oxidase proprotein (SEQ ID NO: 46), this would entail conversionof either all or a subset of T140, T325, S373, T391, and T423 to alanineor another result effective amino acid residue. Conversion of lysineoxidase residues N138, N323, N371, N389, and N421 to glutamine (Q)residues results in loss of enzymatic activity, indicating that thisparticular set of substitutions is not preferred. For the tedanalactamsynthase protein (SEQ ID NO: 41), S188 and S424 could be converted toalanine residues.

[0167] Having constructed apoplastically targeted, glycosylationdeficient lysine oxidase or tedanalactam synthase proteins, it is alsopossible to retain the proteins in the endoplasmic reticulum. This couldbe accomplished by an in frame fusion of DNA sequence encoding thepeptide sequence KDEL to the C-termini of the apoplastically targetedlysine oxidase or tedanalactam synthase coding sequences (Munro andPelham, 1987; Tillmann et al., 1989). It would also similarly bepossible to achieve vacuolar localization via in frame fusions ofvacuolar targeting signals (Bednarek et al., 1990; Neuhaus et al., 1991)to the C-termini of the apoplastically targeted, glycosylation deficientlysine oxidase or tedanalactam synthase proteins.

[0168] To target lysine oxidase enzyme or proenzyme to peroxisomes, theC-terminal peroxisomal targeting sequences could be fused in frame tothe C-terminus of lysine oxidase (Gould et al., 1987; Volokita, 1991).In this example, an amino terminal peroxisomal targeting signal (nPTS)derived from a rice malate dehydrogenase gene (Seq ID NO: 54; SEQ ID NO:55) was fused to the N-terminus of the lysine oxidase proenzyme with sixN-terminal histidine residues. A plant expression cassette consisting ofthe 4as-1 promoter, the rice actin intron, the wheat CAB leader, thenPTS-lysine oxidase fusion gene, and a wheat tahsp 17 3′ polyadenylationsite was constructed in pMON25092. The pMON25092 NotI cassettecontaining the nPTS-lysine oxidase expression cassette was engineeredinto pMON33701 to obtain pMON38800, a plant transformation vectorcontaining the nPTS-Lysine oxidase expression cassette as well aspreviously described neomycin phosphotransferase and tedanalactamsynthase expression cassettes (FIG. 29).

[0169] Transgenic corn expressing both lysine oxidase and tedanalactamsynthase in roots were obtained with pMON38800. Phenotypic analysis ofthis lines indicated that use of the nPTS targeting signal did notalleviate stunting associated with high level lysine oxidase expressionin leaves. However, root specific or enhanced expression of themitochondrial targeted lysine oxidase expression may alleviate stunting.It is also possible that a strategy similar to that described abovecould be used in combination with plant protease insensitive variants oflysine oxidase proenzyme to obtain transgenic maize that are notstunted.

Example 13

[0170] This example illustrates activation of lysine oxidase proproteinby corn rootworm midgut proteases and engineering of improved proenzymevariants.

[0171] The lysine oxidase gene encodes a 69 kDa proenzyme which isN-terminally cleaved when expressed in Trichoderma or Saccharomyces toyield a mature and enzymatically active protein of approximately 60 kDa.To determine if the lysine oxidase −69 (LO69) proenzyme (Seq ID NO: 46)expressed in corn protoplasts can be activated by the proteases presentin the midgut of southern corn rootworm, midguts were dissected fromlate instar larvae and the proteases extracted. Protease activity assaysconfirmed the presence of proteases in this extract. This extract andappropriate controls were incubated for about 22 hours at 25° C. withextracts from protoplasts electroporated with pMON25040 (i.e. theCaMV35S promoter and hsp70 intron fused to the 1.9 kb lysine Is oxidasegene encoding the M_(r) 69,000 proprotein, SEQ ID NO: 45) andnon-electroporated controls and assayed for lysine oxidase activity.These results are summarized in Table 20. TABLE 20 Lysine oxidaseactivity in corn leaf protoplast extracts treated with southern cornrootworm midgut extracts. Sample Treatment Lysine oxidase Sp. Act. FoldDifference¹ LO69 gut extract 1292 U/μg protein 130 LO69 gut extract, 99797 protease inhibitor LO69 BSA 12 1.2 LO69 heated gut extract 10 0control all above 0 NA treatments

[0172] To confirm that the observed increase in lysine oxidase activitywas due to proteolysis of the proprotein, the LO69 and control extractstreated with both active and heated gut extract were analyzed by Westernblot with antibodies directed against lysine oxidase. LO69 extracttreated with the gut extract yielded a band that migrates with the“mature” lysine oxidase of approximately M_(r) 60,000 while LO69extracts treated with heated gut extract yield a much larger band ofapproximately M_(r) 69,000. The Western data thus support our hypothesisthat the CRW gut extracts activate the LO69 proenzyme by proteolysis.

[0173] These results indicate that expression of the LO69 zymogen intransgenic plants may prevent deleterious effects of lysine oxidase onplant growth and development. However, ingestion of LO69 expressing roottissue by CRW is likely to result in activation of lysine oxidase byproteolysis and subsequent insecticidal activity when the tedanalactamsynthase protein is also present. To test the hypothesis thatenzymatically inactive LO69 has insecticidal activity, LO69 proenzyme(LO69) was purified from a heterologous Saccharomyces cerevisiae systemand tested for bioactivity in a diet overlay assay with SCRW (Table 21).The absence of LO activity in the proenzyme sample was confirmed bydirect enzymatic assays just prior to exposure to insects. The resultsof this experiment indicate that SCRW growth is inhibited by ingestionof enzymatically inactive LO69 protoxin, supporting the concept thatenzymatically inactive LO69 could be produced within transgenic plantsto yield control of target insects upon ingestion. TABLE 21 Bioactivityof the LO69 proenzyme when combined with Tedanalactam Synthase (50 kD)against Southern Corn Rootworm Larvae Av. Wght % Sample (ppm)¹ n Surv(mg) SEM² inhibition³ Tris control 18 18 2.6 0.48 50kD (2) 18 18 3.50.46 0 50kD (10)** 18 18 1.5 0.39 39 LO69 (2) 18 17 2.1 0.56 20 LO69(10) 17 17 3.3 0.52 0 LO69 (20) 18 18 2.8 0.51 0 50kD(2)/ LO(2) 18 181.6 0.37 39 50kD(2)/ LO69(2) 18 18 1.5 0.4 41 50kD(2)/LO69(10) 18 180.34 0.03 87 50kD(2)/LO69(20) 18 15 0.59 0.26 77

[0174] Note that the stability of the native LO69 zymogen in planta maybe improved by changing the amino acid sequences in the proproteinregion that are recognized by plant proteases. These sequences can beidentified by obtaining the N-terminal protein sequence of enzymaticallyactive lysine oxidase (i.e. the approximately M_(r) 60,000 proteinproduced by proteolysis of the M_(r) 69,000 proprotein) isolated fromtransgenic plants. For example, mature and enzymatically active lysineoxidase from roots of corn transformed with pMON25040 or pMON30417 isapparently produced by cleavage at the C-terminus of the glu 80 residue.A mutagenized or improved LO69 proenzyme would display greater stabilityin transgenic plants, resulting in lower levels of enzymatically activelysine oxidase and decreased impact of plant growth and/or lysinecontent. The improved LO69 proenzyme would retain amino acid sequencesrecognized by coleopteran midgut proteases, resulting in full activationand biological activity upon ingestion (Table 22). TABLE 22 Design oflysine oxidase proenzyme variants resistant to plant proteases yetsusceptible to target insect midgut proteases Name Sequence Type SEQ IDNO WT1 N-KPALLKEAPRAEEELPPRK-C wild type 46 mut1 N-KPALLGGGGXXXXXXGGGG-Cmutant 56 mut2 N-GGGSGGXXXXXXGGGPPRK-C mutant 57 mut3N-KPGGGGXXXXXXGGGPPRK-C mutant 58

[0175] It is also recognized that amino acid residues lys 68 through lys86 of the lysine oxidase proenzyme (SEQ ID NO: 46) represent a proteasesensitive region in the lysine oxidase proprotein or zymogen. Variantsin this region may yield the desired characteristic of being susceptibleto CRW gut proteases, yet resistant to plant root proteases. Onepreferred embodiment of this invention would be the substitution of thisentire region of nineteen (19) amino acids with a peptide sequence thatrepresents an optimal corn rootworm gut protease cleavage site.Potential representation of this type of amino acid substitutions areshown in SEQ ID. NOS: 56-58. Methods for identifying optimal proteasesubstrates in combinatorial peptide libraries have been identified andcould be employed (Duan and Laursen, 1994). For example, a library ofprotease substrates consisting of a randomized target protease cleavagesite separating a floresence donor and acceptor pair can be immobilizedon a cellulose filter. This filter is then exposed to plant and insectgut proteases; peptides that floresce only in the presence of insectproteases would represent preferred substrates.

[0176] It is finally recognized that the entire lysine oxidase proenzymesequence (SEQ ID NO: 46) extending from amino acid residues 1 (met) to87 (val) may be modified to encode a variant with the desired propertyof being resistant to activation by corn root proteases yet sensitive toactivation by corn rootworm gut proteases. This region could bemutagenized by either site-directed or random mutagenesis techniquesfamiliar to those skilled in the art (Kunkel, 1985; Spee et al, 1993;Muhlrad et al, 1992). The population of mutagenized lysine oxidaseproprotein expressing clones expressed in Saccharomyces cerevisiae couldpotentially be screened via exposure to plant and insect proteasesfollowed by lysine oxidase enzymatic assays to identify the variant withthe desired protease activation properties. Finally, the improved lysineoxidase zymogen could be targeted to plastids, mitochondria, theapoplastic space, or the vacuole as described above.

[0177] The lysine oxidase proenzyme sequence extending from amino acids1 to 87 (SEQ ID NO: 46) may also be fused to proteins other than lysineoxidase to create other zymogens that would be activated upon insectingestion. The resultant chimeric protein could then be activated bycoleopteran or lepidopteran midgut proteases to yield enzymaticallyactive, insecticidal proteins.

[0178] All of the compositions and methods disclosed and claimed hereincan be made and executed without undue experimentation in light of thepresent disclosure. While the compositions and methods of this inventionhave been described in terms of preferred embodiments, it will beapparent to those of skill in the art that variations may be applied tothe compositions and methods and in the steps or in the sequence ofsteps of the methods described herein without departing from theconcept, spirit and scope of the invention. All such similar substitutesand modifications apparent to those skilled in the art are deemed to bewithin the spirit, scope and concept of the invention as defined by theappended claims.

[0179] In view of the above, it will be seen that the several advantagesof the invention are achieved and other advantageous results attained.

[0180] As various changes could be made in the above methods andcompositions without departing from the scope of the invention, it isintended that all matter contained in the above description and shown inthe accompanying drawings shall be interpreted as illustrative and notin a limiting sense.

[0181] References

[0182] The following references, to the extent that they provideexemplary procedural or other details supplementary to those set forthherein, are specifically incorporated herein by reference.

[0183] The following references, to the extent that they provideexemplary procedural or other details supplementary to those set forthherein, are specifically incorporated herein by reference.

[0184] Armstrong, C. L. et al. Plant Cell Rep. 9: 335-339 (1990).

[0185] Bagdasarian, M. et al. Gene 16: 237-247 (1981).

[0186] Barry, G. F. and Kishore, G. M. “Glyphosate tolerant plants.”U.S. Pat. No. 5,463,175 (1995).

[0187] Barton, K. et al. Plant Physiol. 85: 1103-1109 (1987).

[0188] Bechtold, N. et al. C. R. Acad. Sci. Parish Life Sciences 316:1194-1199 (1993).

[0189] Bednarek, S. Y. et al. Plant Cell 2: 1145-1155 (1990).

[0190] Bevan, M. et al. Nature 304: 184 (1983).

[0191] Boutry, M. et al. Nature 328: 340-342 (1987).

[0192] Bright, H. J. and Porter, D. J. T. in The Enzymes, Vol.12B:421-505 (1975).

[0193] Brown, S. M. and Santino, C. G. “Enhanced expression in plants.”U.S. Pat. No. 5,424,412 (1995).

[0194] Corbin, D. R. et al. Appl. Environ. Microbiol. 60 (12): 4239-4244(1994).

[0195] Cornelissen, B. J. C. et al. EMBO J. 5: 37-40 (1986).

[0196] Cronan Jr., J. M. and Cardellina, J. H. Natural Products Lett. 5:85-88 (1994).

[0197] Ditta, G. et al. Proc. Natl. Acad. Sci. U.S.A. 77: 7347-7351(1980).

[0198] Duan, Y. and Laursen, R. A. Anal. Biochem. 216: 431-438 (1994).

[0199] Fedoroff, N. et al. Cell 35: 235-242 (1983).

[0200] Feinberg, A. P. and Vogelstein, B. Anal. Biochem. 132: 6-13(1983).

[0201] Fischhoff, D. A. and Perlak, F. J. “Synthetic plant genes andmethod for preparation.” European Patent Application, Publication Number0 385 962 (1990).

[0202] Fischhoff, D. A. et al. Bio/Technology 5: 807-813 (1987).

[0203] Frischauf, A. M. et al. Methods Enzymol. 153: 103-115 (1987).

[0204] Frohman, M. A. et al. Proc. Natl. Acad. Sci. U.S.A. 85: 8998-9002(1988).

[0205] Fromm, M. E. et al. Bio/Technology 8: 833-839 (1990).

[0206] Fromm, M. E. et al. Nature 319: 791-793 (1986).

[0207] Gallo Methods Enzymol. 71: 665-667 (1981).

[0208] Gould et al. J. Cell Biol. 105: 2923-2931 (1987).

[0209] Halpin, C. and Ryan, M. “Expression of self-processingpolyproteins in transgenic plants”. International Patent ApplicationNumber WO 95/17514 (1995).

[0210] Herrera-Estrella, L. et al. Nature 303: 209 (1983).

[0211] Hills, T. M. and Peters, D. C. J. Econ. Entomol. 64: 764-765(1971).

[0212] Hope, et al. Biochem. J. 105: 663-667 (1967).

[0213] Horsch, R. B. et al. Science 227: 1229-1231 (1985).

[0214] Horton, R. M. et al. Gene 77: 61-68 (1989).

[0215] Klee, H. J. et al. Bio/Technology 3: 637-642 (1985).

[0216] Knauf, V. C. and Nester, E. Plasmid 8: 43-54 (1982).

[0217] Knight, S. G. J. Bacteriol. 55: 401-407 (1948).

[0218] Koehler, S. M. and Ho, T. H. Plant Cell 2(8): 769-783 (1990).

[0219] Kreig, A. et al. Pathotyp. Z. Ang. Ent. 96: 500-508 (1983).

[0220] Kunkel, T. A. Proc. Natl. Acad. Sci. USA 82:488-492 (1985)

[0221] Kusakabe, H. et al. J. Biol. Chem. 256: 976-981 (1980).

[0222] Kusakabe, H. et al. Azric. Biol. Chem. 43: 337-343 (1979).

[0223] Lam et al. “Promoter enhancer element for gene expression inplant roots”. U.S. Pat. No. 5,023,179 (1991).

[0224] Lee, C. C. et al. Science 239: 1288-1291 (1988).

[0225] Matsudaira, P. J. Biol. Chem. 261: 10035-10038 (1987).

[0226] McElroy, D. et al. Plant Cell 2: 163-171 (1990).

[0227] Muhlrad et al. Yeast 8:79-82 (1992).

[0228] Munro, S. and Pelham, H. R. B. Cell 48: 899-907 (1987).

[0229] Neuhaus, J-M. et al. Proc. Natl. Acad. Sci. USA 88:10362-10366(1991).

[0230] Niedermann, D. M. and Lerch, K. J. Biol. Chem. 265: 17246-17251(1990).

[0231] Perlak, F. J. et al. Bio/technology 8: 939-943 (1990).

[0232] Rogers, S. G. “Promoter for transgenic plants”. U.S. Pat. No.5,378,619 (1995).

[0233] Russell, D. A. et al. Plant Cell Reports 13: 24-27 (1993).

[0234] Schilperoort et al. EPO publication 0 120 516.

[0235] Spree, J. H. et al. Nuc. Acid. Res. 21:77-778 (1993).

[0236] Stumpf, P. K. and Green, D. E. J. Biol. Chem. 153: 387 (1944).

[0237] Tillmann, U. et al. EMBO J. 8(9): 2463-2467 (1989).

[0238] Vaeck, M. et al. Nature 328: 33-37 (1987).

[0239] Volokita, M. Plant J. 1: 361-366 (1991)

[0240] White, J. A. and Scandalios Proc. Nat. Acad. Sci. U.S.A.86:3534-3538 (1989).

[0241] Winter, J. et al. Mol. Gen. Genet. 221(2): 315-319 (1988).

[0242] Xu, Y. et al. Plant Mol. Biol. 27: 237-248 (1995).

[0243] Yamamoto, Y. et al. Plant Cell 3: 371-382 (1991).

1 58 1 22 PRT Artificial Sequence Synthetic Peptide 1 Asp Ala Pro ProGln Pro Pro Lys Glu Asp Glu Leu Val Glu Leu Ile 1 5 10 15 Leu Gln AsnLeu Ala Arg 20 2 20 DNA Artificial Sequence Synthetic Oligonucleotide 2tcrtcytcyt tnggnggytg 20 3 13 PRT Artificial Sequence Synthetic Peptide3 Gly Leu Asn Leu His Pro Thr Gln Ala Asp Ala Ile Arg 1 5 10 4 23 DNAArtificial Sequence Synthetic Oligonucleotide 4 atngcrtcng cytgngtnggrtg 23 5 23 DNA Artificial Sequence Synthetic Oligonucleotide 5cayccnacnc argcngaygc nat 23 6 20 DNA Artificial Sequence SyntheticOligonucleotide 6 aayctncayc cnacncargc 20 7 20 DNA Artificial SequenceSynthetic Oligonucleotide 7 aayttrcayc cnacncargc 20 8 9 PRT ArtificialSequence Synthetic Peptide 8 Lys Gln Gln Ala Phe Gly Tyr Tyr Lys 1 5 920 DNA Artificial Sequence Synthetic Oligonucleotide 9 aarcarcargcnttyggnta 20 10 20 DNA Artificial Sequence Synthetic Oligonucleotide 10cargcnttyg gntaytayaa 20 11 12 PRT Artificial Sequence Synthetic Peptide11 Tyr Pro Ser Tyr Asn Xaa Asp Asp Thr Gly Glu Ala 1 5 10 12 763 DNAArtificial Sequence Synthetic Polynucleotide 12 aagcagcagg cgtgggtattacaaagagaa gcttgctgag gacttcgaca aagggttcga 60 tgagctcatg ctcgtcgacgacatgaccac tcgagagtac ttgaagcgag gcgggccgaa 120 gggagaggcg cccaagtatgactttttcgc catccagtgg atggagacac aaaacactgg 180 gacaaacctg tttgatcaggccttttctga aagcgtcatc gactcgtttg actttgacaa 240 cccgacaaag cccgaatggtactgcatcga gggaggaaca tcgcttttgg tggacgccat 300 gaaagaaacc cttgtccacaaggtacagaa caacaagaga gttgatgcca tttccattga 360 cttggacgct ccggatgatgggaacatgtc ggtcaggata ggcggaaagg atcactccgg 420 atatagcacc gtcttcaacaccaccgctct gggctgcctt gaccgcatgg atctgcgtgt 480 ctcaacttgc accctactcaggcagatgcc attcgatgtt tgcactatga caactcgacc 540 aaggtggctc tcaagtttactacccgtggt ggatcaagga ctgtggcatc acttgcggtg 600 gcgcggcctc gactgatctacctctacgaa cttgcgttta cccatcatac aacttggacg 660 atactggtga ggctgttctgcttgcctcat acacttggtc tcaagatgca actcgcattg 720 gatcgttggt gaaggacgctccaccacacc ccccaaagaa gac 763 13 23 DNA Artificial Sequence SyntheticOligonucleotide 13 acctctacga acttgcgttt acc 23 14 23 DNA ArtificialSequence Synthetic Oligonucleotide 14 caactcgcat tggatcgttg gtg 23 152700 DNA Artificial Sequence Synthetic Polynucleotide 15 gatctaaccacggcttttgc gctccaggcc gctcccaccg catgcaggca ttccatccca 60 accctcaaatgagtctagcc ttcagccttc acctgcaagt ggctggcggg atgtgttcgg 120 gacttcatgcagctcaagac tctgagcctc gctgatgatg aggggattca agacatgcat 180 ttcagctttggtgataagag ccaatagtgt ttgctgctca tgttgtctgt gctttctgtg 240 ccgcttctgtgccgttatcg cctgttttat agcgtcagcc aagccaatca gtctcctccc 300 gctggaatccctcccgtgtc atttttctcc ccgttacgca attcttcctt aatcgatact 360 actatacagtatgatggaga gcttttactg gtgcccactt tgtggcaatg ctattgatgt 420 ctttcaagtcagagctgagc acggaaatcg atagcctgac ctctaacggc tgtcggtagc 480 tgaaaggggatgagagcgga ggcggttaat tcagctaggt attgattaag ggaactggca 540 gcttgtgttcacgtaggctc tgaataagat ataaataagg agaggaaagg ctacgcaatc 600 gaagtaaacggctaccatcg ccatcttctc atcatagcta tcccgttact atatttgcaa 660 acatggacaatgttgacttt gctgaatctg tccgaacccg ctgggcgagg cgactcattc 720 gtgagaaggtcgccaaggaa ctcaacattc taaccgaaag acttggtgag gtgcccggca 780 tccctcctccaaatgaaggc aggttcctgg gcggcggcta ctctcacgac aatctaccgt 840 ctgatcccctctattccagc attaagccgg ctcttctaaa ggaggctcct cgagcagaag 900 aggaactgccgcctcgaaag gtgtgcatcg taggcgctgg tgtttccggc ctctacatag 960 ccatgattttggatgatttg aaaatcccaa atctcactta cgacatcttc gaatccagtt 1020 ccagaactggtggccgtttg tatacgcacc atttcaccga cgccaagcat gactattacg 1080 acattggtgctatgcgatat cctgacatcc ccagcatgaa acgtaccttt aacctgttta 1140 aacgtactgggatgcctctc atcaaatatt accttgatgg cgagaatacc cctcagctgt 1200 acaataatcacttcttcgcc aagggcgtgt cggaccccta tatggtgagc gtggccaatg 1260 gcggcaccgtgccagatgat gttgtcgata gtgttggaga gaagttacaa caggctttcg 1320 gttattacaaagagaagctt gctgaggact tcgacaaagg gttcgatgag ctcatgctcg 1380 tcgacgacatgaccactcga gagtacttga agcgaggcgg gccgaaggga gaggcgccca 1440 agtatgactttttcgccatc cagtggatgg agacacaaaa cactgggaca aacctgtttg 1500 atcaggccttttctgaaagc gtcatcgact cgtttgactt tgacaacccg acaaagcccg 1560 aatggtactgcatcgaggga ggaacatcgc ttttggtgga cgccatgaaa gaaacccttg 1620 tccacaaggtacagaacaac aagagagttg atgccatttc cattgacttg gacgctccgg 1680 atgatgggaacatgtcggtc aggataggcg gaaaggatca ctccggatat agcaccgtct 1740 tcaacaccaccgctctgggc tgccttgacc gcatggatct gcgtggtctc aacttgcacc 1800 ctactcaggcagatgccatt cgatgtttgc actatgacaa ctcgaccaag gtggctctca 1860 agtttagctacccgtggtgg atcaaggact gtggcatcac ttgcggtggc gcggcctcga 1920 ctgatctacctctacgaact tgcgtttacc catcatacaa cttggacgat actggtgagg 1980 ctgttctgcttgcctcatac acttggtctc aagatgcaac tcgcattgga tcgttggtga 2040 aggacgctccaccacagccg cccaaggagg atgagcttgt cgagctgatc ctgcagaacc 2100 tagcccgcctgcacgctgag catatgacct acgagaagat taaggaggct tacacgggcg 2160 tatatcacgcctattgctgg gctaatgatc ccaatgtcgg tggtgctttc gccctcttcg 2220 gtcccggccagttcagcaat ctgtatccat acctgatgcg gccagcggcg ggcggcaagt 2280 tccatatcgtcggagaggca tctagtgtgc atcacgcctg gatcataggg tctttggaga 2340 gcgcttacaccgctgtgtac cagttcttgt acaagtacaa gatgtgggat tacttgaggt 2400 tgttgttggagcgctggcag tatggtctcc aggagttaga gacggggaag cacggtacgg 2460 ctcatttgcagtttattcta ggttcacttc ccaaggagta ccaggtgaag atttaaagcg 2520 aaagaggtactacggcatgg agacaatttt gggtagagat tctagtattc cagcagtttc 2580 atagaaagtgtgatgtttgt tagtcccact ttgagtctct gttcgtctga aagtgcctac 2640 tatgacccggtgattagtat aacagaattt gtcattctca tcagccataa accgaggtca 2700 16 22 DNAArtificial Sequence Synthetic Oligonucleotide 16 catgtcgtcg acgagcatgagc 22 17 23 DNA Artificial Sequence Synthetic Oligonucleotide 17catcgaaccc tttgtcgaag tcc 23 18 25 DNA Artificial Sequence SyntheticOligonucleotide 18 cagcaagctt ctctttgtaa taccc 25 19 274 DNA ArtificialSequence Synthetic Polynucleotide 19 actatacgac attggtgcta tgcgatatcctgacatcccc agcatgaaac gtacctttaa 60 cctgtttaaa cgtactggga tgcctctcatcaaatattac cttgatggcg agaatacccc 120 tcagctgtac aataatcact tcttcgccaagggcgtgtcg gacccctata tggtgagcgt 180 ggccaatggc ggcaccgtsc cagatgatgttngtcgatag tgttggagag aagttacaac 240 aggctttcgg gtattacaaa gagaagcttgctga 274 20 31 DNA Artificial Sequence Synthetic Oligonucleotide 20gtcgaagtcc tcagccagct tctctttgta a 31 21 18 DNA Artificial SequenceSynthetic Oligonucleotide 21 catgctgggg atgtcagg 18 22 262 DNAArtificial Sequence Synthetic Polynucleotide 22 accatcgcca tcttctcatcatagctatcc cgttactata tctgcaaaca tggacaatgt 60 tgactttgct gaatctgtccgaacccgctg ggcgaggcga ctcattcgtg agaaggtcgc 120 caaggaactc aacattctaaccgaaagact tggtgaggtg cccggcatcc ctcctccaaa 180 tgaaggcagg ttcctgggcggcggctactc tctcgacaat ctaccgcctg atcccctcta 240 ttccagcatt aagccggctc tt262 23 26 DNA Artificial Sequence Synthetic Oligonucleotide 23garcaraaya ayttyttyaa ycaygc 26 24 20 PRT Artificial Sequence SyntheticPeptide 24 Val Val Val Leu Glu Gln Asn Asn Phe Phe Asn His Ala Gly SerSer 1 5 10 15 Asn Asp Leu Ala 20 25 21 DNA Artificial Sequence SyntheticOligonucleotide 25 atgtayacng arcaytayat g 21 26 13 PRT ArtificialSequence Synthetic Peptide 26 Thr Met Tyr Thr Glu Asp Tyr Met Ala AspLeu Ala Lys 1 5 10 27 20 DNA Artificial Sequence SyntheticOligonucleotide 27 ggngcraayt graaccacat 20 28 15 PRT ArtificialSequence Synthetic Peptide 28 Gly Thr Ile Phe Pro Ser Met Trp Phe GlnPhe Ala Pro Asp Lys 1 5 10 15 29 623 DNA Artificial Sequence SyntheticPolynucleotide 29 atgtatacgg aggactacat ggccgatctt gccaaggaag ccttggccctctgggatgat 60 cttgagagag attccggtac gccactgcga tggatgagcg gcctcctcaactttggcgat 120 aaggactatg gcggcgatac acccgaagga accttgttgg ggccaattgcgaacctggac 180 cgcctgggaa tgacttatca agagttatct gctaaggaga ttgaggcacgctacccgttc 240 aagaacctcg accctaagta cattggtctc ttcgcgccag acaatggcgtcatcaatgtc 300 cagcttctgt tgaggacgct gtataaatta tcactggact atggtgccactgcgaaacag 360 cataccaaag tccaggctat taagccttct aatcattctc attacgcctgggatgttcac 420 gctattcgtc atgagaccga agccgctgtc ttcaaggcaa agaagatcattatcgcctct 480 ggtgcttacg tgaaccatgt tctcaagccg agcttcgaca tttctctcgatctcgacatc 540 tgagaaatgg tgttttctta ctttaactgc aatgcaggac ccaaaggaacaatattcccc 600 agcatgtggt tccaattcgc ccc 623 30 11 PRT ArtificialSequence Synthetic Peptide 30 Leu Gly Met Thr Tyr Gln Glu Met Ser AlaLys 1 5 10 31 27 DNA Artificial Sequence Synthetic Peptide 31 ccggaattccttggcaagat cggccat 27 32 19 DNA Artificial Sequence Synthetic Peptide 32cctccgtata cattgttcg 19 33 381 DNA Artificial Sequence SyntheticPolynucleotide 33 gaattcggct tctactacta ctaggccacg cgtcgactag tacggggggggggggggtgg 60 gggtgacatc acgttgtttc agtgctggat ataggttcct cctagagtttacctattgag 120 acagatactt caatcacatt ctctaggata tcgaatcaaa ccgaaaacacttgcttcaga 180 atcccctaaa catggcagac gaaatctacg atgttgtcgt catcggcggcggcccaattg 240 gattggcagc tgcctatgaa gcagccaagg agggtgccaa agtcgttgttctcgagcaaa 300 acaatttctt caaccatgct gggagctcta acgatttggc tcggatgtttcgaacaatgt 360 atacggagga agccgaattc c 381 34 31 DNA Artificial SequenceSynthetic Peptide 34 ccggaattca tggccgatct tgccaaggaa g 31 35 1426 DNAArtificial Sequence Synthetic Polynucleotide 35 gaattcggct tccggaattcatggccgatc ttgccaagga agccttggcc ctctgggatg 60 atcttgagag agattccggtacgccactgc gatggatgag cggcctcctc aactttggcg 120 ataaggacta tggcggcgatacacccgaag gaaccttgtt ggggccaatt gcgaacctgg 180 accgcctggg aatgacttatcaagagttat ctgctaagga gattgaggca cgctacccgt 240 tcaagaacct cgaccctaagtacatcggtc tcttcgcgcc agacaatggg ctcatcaatg 300 tccagcttct gttgaggacgctgtataaat tatcactgga ctatggtgcc actgcgaaac 360 agcataccaa agtccaggctattaagcctt ctaatcattc tcattacgcc tgggatgttc 420 acgctattcg tcatgagaccgaagccgctg tcttcaaggc aaagaagatc attatcgcct 480 ctggtgctta cgtgaaccatgttctcaagc cgagcttcga catttctctc gatctcgaca 540 tctgggaaat ggtgttttcttactttaact gcaatgcagg acccaaagga acaatattcc 600 ccagcatgtg gttccagtttgcgcctgata agaacggcag atcacagctc ttctatggct 660 ttccagcact tccatggggccctccaaatc ttgctcgtat tgctatggat gcggccacca 720 ggcggatcaa ggatcccaacgagagactta caagcactat taacccggag gatattgctg 780 atacgcaaga gtttatccgcaatcattgtg tcaacgttga tcctaccatt cctgcgttga 840 catcgagttg cctgcagaccaatgtgtttg acaacatgtt tgttctggac tttgtccctg 900 aaaaatatct gaacggcggagccaaagaca gtgtagtcgt cttcacagcc ggatgggcca 960 tgaagttcgt gccaatgataggaaaggcac tcgctgacat ggcactcaag ggaagctctc 1020 catatgcgcg caaagaatttgccatcaccc gcacagattc agcgaccggg aagggcatca 1080 ttgtggaagg tggatcagagaaccgatcgg ttaagagcag cgcttttgtc ttctactcac 1140 caggcatccg gttcttcgtttgccggcttc cataacactg cacggcaata gaagaaagtg 1200 aataggggta agcgggcgggataggatatc tgtggaacac acaatgagaa gtgaccaaga 1260 tcgctgttga gaatacgccaaagcatacta tagcttgtag gtgttgctat ctggtctaca 1320 gtgttgcaaa gatgcataaataggtgaaaa agaattgatg aggtatatga atcctcagta 1380 aaaaaaaaaa aaaaaaatcgatgtcgactc gagtcaagcc gaattc 1426 36 27 DNA Artificial SequenceSynthetic Oligonucleotide 36 gggagatctc catggcagac gaaatct 27 37 31 DNAArtificial Sequence Synthetic Oligonucleotide 37 ggctttccag cacttccttggggccctcca a 31 38 31 DNA Artificial Sequence Synthetic Oligonucleotide38 ttggagggcc ccaaggaagt gctggaaagc c 31 39 31 DNA Artificial SequenceSynthetic Oligonucleotide 39 cccaagcttg aattcacttt cttctattgc c 31 401385 DNA Artificial Sequence Synthetic Polynucleotide 40 gggagatctccatggcagac gaaatctacg atgttgtcgt catcggcggc ggcccaattg 60 gattggcagctgcctatgaa gcagccaagg agggtgccaa agtcgttgtt ctcgagcaaa 120 acaatttcttcaaccatgct gggagctcta acgatttggc tcggatgttt cgaacaatgt 180 atacggaggattatatggcc gatcttgcca aggaagcctt ggccctctgg gatgatcttg 240 agagagattccggtacgcca ctgcgatgga tgagcggcct cctcaacttt ggcgataagg 300 actatggcggcgatacaccc gaaggaacct tgttggggcc aattgcgaac ctggaccgcc 360 tgggaatgacttatcaagag ttatctgcta aggagattga ggcacgctac ccgttcaaga 420 acctcgaccctaagtacatt ggtctcttcg cgccagacaa tgggctcatc aatgtccagc 480 ttctgttgaggacgctgtat aaattatcac tggactatgg tgccactgcg aaacagcata 540 ccaaagtccaggctattaag ccttctaatc attctcatta cgcctgggat gttcacgcta 600 ttcgtcatgagaccgaagcc gctgtcttca aggcaaagaa gatcattatc gcctctggtg 660 cttacgtgaaccatgttctc aagccgagct tcgacatttc tctcgatctc gacatctggg 720 aaatggtgttttcttacttt aactgcaatg caggacccaa aggaacaata ttccccagca 780 tgtggttccagtttgcgcct gataagaacg gcagatcaca gctcttctat ggctttccag 840 cacttccttggggccctcca aatcttgctc gtattgctgt ggatgcggcc accaggcgga 900 tcaaggatcccaacgagaga cttacaagca ctattaaccc ggaggatatt gctgatacgc 960 aagagtttatccgcaatcat tgtgtcaacg ttgatcctac cattcctgcg ttgacatcga 1020 gttgcctgcagaccaatgtg tttgacaaca tgtttgttct ggactttgtc cctgaaaaat 1080 atctgaacggcggagccaaa gacagtgtag tcgtcttcac agccggatgg gccatgaagt 1140 tcgtgccaatgataggaaag gcactcgctg acatggcact caagggaagc tctccatatg 1200 cgcgcaaagaatttgccatc acccgcacag attcagcgac cgggaagggc atcattgtgg 1260 aaggtggatcagagaaccga tcggttaaga gcagcgcttt tgtcttctac tcaccaggca 1320 tccggttcttcgtttgccgg cttccataac actgcacggc aatagaagaa agtgaattca 1380 agctt 138541 445 PRT Artificial Sequence Synthetic Polypeptide 41 Met Ala Asp GluIle Tyr Asp Val Val Val Ile Gly Gly Gly Pro Ile 1 5 10 15 Gly Leu AlaAla Ala Tyr Glu Ala Ala Lys Glu Gly Ala Lys Val Val 20 25 30 Val Leu GluGln Asn Asn Phe Phe Asn His Ala Gly Ser Ser Asn Asp 35 40 45 Leu Ala ArgMet Phe Arg Thr Met Tyr Thr Glu Asp Tyr Met Ala Asp 50 55 60 Leu Ala LysGlu Ala Leu Ala Leu Trp Asp Asp Leu Glu Arg Asp Ser 65 70 75 80 Gly ThrPro Leu Arg Trp Met Ser Gly Leu Leu Asn Phe Gly Asp Lys 85 90 95 Asp TyrGly Gly Asp Thr Pro Glu Gly Thr Leu Leu Gly Pro Ile Ala 100 105 110 AsnLeu Asp Arg Leu Gly Met Thr Tyr Gln Glu Leu Ser Ala Lys Glu 115 120 125Ile Glu Ala Arg Tyr Pro Phe Lys Asn Leu Asp Pro Lys Tyr Ile Gly 130 135140 Leu Phe Ala Pro Asp Asn Gly Leu Ile Asn Val Gln Leu Leu Leu Arg 145150 155 160 Thr Leu Tyr Lys Leu Ser Leu Asp Tyr Gly Ala Thr Ala Lys GlnHis 165 170 175 Thr Lys Val Gln Ala Ile Lys Pro Ser Asn His Ser His TyrAla Trp 180 185 190 Asp Val His Ala Ile Arg His Glu Thr Glu Ala Ala ValPhe Lys Ala 195 200 205 Lys Lys Ile Ile Ile Ala Ser Gly Ala Tyr Val AsnHis Val Leu Lys 210 215 220 Pro Ser Phe Asp Ile Ser Leu Asp Leu Asp IleTrp Glu Met Val Phe 225 230 235 240 Ser Tyr Phe Asn Cys Asn Ala Gly ProLys Gly Thr Ile Phe Pro Ser 245 250 255 Met Trp Phe Gln Phe Ala Pro AspLys Asn Gly Arg Ser Gln Leu Phe 260 265 270 Tyr Gly Phe Pro Ala Leu ProTrp Gly Pro Pro Asn Leu Ala Arg Ile 275 280 285 Ala Val Asp Ala Ala ThrArg Arg Ile Lys Asp Pro Asn Glu Arg Leu 290 295 300 Thr Ser Thr Ile AsnPro Glu Asp Ile Ala Asp Thr Gln Glu Phe Ile 305 310 315 320 Arg Asn HisCys Val Asn Val Asp Pro Thr Ile Pro Ala Leu Thr Ser 325 330 335 Ser CysLeu Gln Thr Asn Val Phe Asp Asn Met Phe Val Leu Asp Phe 340 345 350 ValPro Glu Lys Tyr Leu Asn Gly Gly Ala Lys Asp Ser Val Val Val 355 360 365Phe Thr Ala Gly Trp Ala Met Lys Phe Val Pro Met Ile Gly Lys Ala 370 375380 Leu Ala Asp Met Ala Leu Lys Gly Ser Ser Pro Tyr Ala Arg Lys Glu 385390 395 400 Phe Ala Ile Thr Arg Thr Asp Ser Ala Thr Gly Lys Gly Ile IleVal 405 410 415 Glu Gly Gly Ser Glu Asn Arg Ser Val Lys Ser Ser Ala PheVal Phe 420 425 430 Tyr Ser Pro Gly Ile Arg Phe Phe Val Cys Arg Leu Pro435 440 445 42 2093 DNA Artificial Sequence Synthetic Polynucleotide 42atatgtcgca ttctggacat tctacggtat cattatttgt ggcgcagtgg tttatacgac 60tcaattgagt attatattaa gccgacattc cgaaggtctt ctctatcgcc acatcacgtt 120gtttcagtgc tggatatagg ttcctcctag agtttaccta ttgagacaga tacttcaatc 180acattctcta ggatatcgaa tcaaaccgaa aacacttgct tcagaatccc ctaaacatgg 240cagacgaaat ctacgatgtt gtcgtcatcg gcggcggccc aattggattg gcagctgcct 300atgaagcagc caaggagggt gccaaagtcg ttgttctcga gcaaaacaat ttcttcaacc 360atgctgggag ctctaacgat ttggctcgga tgtttcgaac aatgtgagtt atttttttgt 420cttttttctt actctcgttt tcacagacac agctaatcat ccgatcaggt atacggagga 480ttatatggcc gatcttgcca aggaagcctt ggccctctgg gatgatcttg agagagattc 540cggtacgcca ctgcgatgga tgagcggcct cctcaacttt ggcgataagg actatggcgg 600cgatacaccc gaaggtatga aatcctccca caataatatg ggttttggcg cccttgtctc 660acgatttcaa caggaacctt gttggggcca attgcgaacc tggaccgcct gggaatgact 720tatcaagagt gtaagttgtg gcatgtatgc gaacgacggt atgccctcga gtgctaatcc 780atcgtctcac agtatctgct aaggagattg aggcacgcta cccgttcaag aacctcgacc 840ctaagtacat tggtctcttc gcgccagaca atgggctcat caatgtccag cttctgttga 900ggacgctgta taaattatca ctggactatg gtgccactgc gaaacagcat accaaagtcc 960aggctattaa gccttctaat cattctcatt acgcctggga tgttcacgct attcgtcatg 1020agaccgaagc cgctgtcttc aaggcaaaga agatcattat cgcctctggt gcttacgtga 1080accatgttct caagccgagc ttcgacattt ctctcgatct cgacatctgg gaaatggtgt 1140tttcttactt taactgcaat gcaggaccca aaggaacaat attccccagt acgtggattg 1200atccatttct ctcgtgagtt ggaggtgtat gagctaactc ccatcaacta ggcatgtggt 1260tccagtttgc gcctgataag aacggcagat cacagctctt ctatggcttt ccagcacttc 1320catggggccc tccaaatctt gctcgtattg ctgtggatgc ggccaccagg cggatcaagg 1380atcccaacga gagacttaca agcactatta acccggagga tattgctgat acgcaagagt 1440ttatccgcaa tcattgtgtc aacgttgatc ctaccattcc tgcgttgaca tcgagttgcc 1500tgcagaccaa tgtgtttggt gcgtatattc atatggatgg attgacaagg aaacttactg 1560attcggctta tagacaacat gtttgttctg gactttgtcc ctgaaaaata tctgaacggc 1620ggagccaaag acagtgtagt cgtcttcaca gccggatggg ccatgaagtt cgtgccaatg 1680ataggaaagg cactcgctga catggcactc aagggaagct ctccatatgc gcgcaaagaa 1740tttgccatca cccgcacaga ttcagcgacc gggaagggca tcattgtgga aggtggatca 1800gagaaccgat cggttaagag cagcgctttt gtcttctact caccaggcat ccggttcttc 1860gtttgccggc ttccataaca ctgcacggca atagaagaaa gtgaataggg ggtaagcagg 1920cgggatagga tatctgtgga acacacaatg agaagtgacc aagatcgctg ttgagaatac 1980gcaaagcata ctatagcttg taggtgttgc tatctggtct acagtgttgc aaagatgcat 2040aaataggtga aaaagaattg atgaggtata tgaatcctca gtaatcttga gcc 2093 43 35DNA Artificial Sequence Synthetic Oligonucleotide 43 ttgcaaaccatggacaatgt tgactttgct gaatc 35 44 33 DNA Artificial Sequence SyntheticOligonucleotide 44 gccgtagtac cgaattctta ttaaatcttc acc 33 45 1883 DNAArtificial Sequence Synthetic Polynucleotide 45 ttgcaaacca tggacaatgttgactttgct gaatctgtcc gaacccgctg ggcgaggcga 60 ctcattcgtg agaaggtcgccaaggaactc aacattctaa ccgaaagact tggtgaggtg 120 cccggcatcc ctcctccaaatgaaggcagg ttcctgggcg gcggctactc tcacgacaat 180 ctaccgtctg atcccctctattccagcatt aagccggctc ttctaaagga ggctcctcga 240 gcagaagagg aactgccgcctcgaaaggtg tgcatcgtag gcgctggtgt ttccggcctc 300 tacatagcca tgattttggatgatttgaaa atcccaaatc tcacttacga catcttcgaa 360 tccagttcca gaactggtggccgtttgtat acgcaccatt tcaccgacgc caagcatgac 420 tattacgaca ttggtgctatgcgatatcct gacatcccca gcatgaaacg tacctttaac 480 ctgtttaaac gtactgggatgcctctcatc aaatattacc ttgatggcga gaatacccct 540 cagctgtaca ataatcacttcttcgccaag ggcgtgtcgg acccctatat ggtgagcgtg 600 gccaatggcg gcaccgtgccagatgatgtt gtcgatagtg ttggagagaa gttacaacag 660 gctttcggtt attacaaagagaagcttgct gaggacttcg acaaagggtt cgatgagctc 720 atgctcgtcg acgacatgaccactcgagag tacttgaagc gaggcgggcc gaagggagag 780 gcgcccaagt atgactttttcgccatccag tggatggaga cacaaaacac tgggacaaac 840 ctgtttgatc aggccttttctgaaagcgtc atcgactcgt ttgactttga caacccgaca 900 aagcccgaat ggtactgcatcgagggagga acatcgcttt tggtggacgc catgaaagaa 960 acccttgtcc acaaggtacagaacaacaag agagttgatg ccatttccat tgacttggac 1020 gctccggatg atgggaacatgtcggtcagg ataggcggaa aggatcactc cggatatagc 1080 accgtcttca acaccaccgctctgggctgc cttgaccgca tggatctgcg tggtctcaac 1140 ttgcacccta ctcaggcagatgccattcga tgtttgcact atgacaactc gaccaaggtg 1200 gctctcaagt ttagctacccgtggtggatc aaggactgtg gcatcacttg cggtggcgcg 1260 gcctcgactg atctacctctacgaacttgc gtttacccat catacaactt ggacgatact 1320 ggtgaggctg ttctgcttgcctcatacact tggtctcaag atgcaactcg cattggatcg 1380 ttggtgaagg acgctccaccacagccgccc aaggaggatg agcttgtcga gctgatcctg 1440 cagaacctag cccgcctgcacgctgagcat atgacctacg agaagattaa ggaggcttac 1500 acgggcgtat atcacgcctattgctgggct aatgatccca atgtcggtgg tgctttcgcc 1560 ctcttcggtc ccggccagttcagcaatctg tatccatacc tgatgcggcc agcggcgggc 1620 ggcaagttcc atatcgtcggagaggcatct agtgtgcatc acgcctggat catagggtct 1680 ttggagagcg cttacaccgctgtgtaccag ttcttgtaca agtacaagat gtgggattac 1740 ttgaggttgt tgttggagcgctggcagtat ggtctccagg agttagagac ggggaagcac 1800 ggtacggctc atttgcagtttattctaggt tcacttccca aggagtacca ggtgaagatt 1860 taataagaat tcggtactacggc 1883 46 617 PRT Artificial Sequence Synthetic Polypeptide 46 Met AspAsn Val Asp Phe Ala Glu Ser Val Arg Thr Arg Trp Ala Arg 1 5 10 15 ArgLeu Ile Arg Glu Lys Val Ala Lys Glu Leu Asn Ile Leu Thr Glu 20 25 30 ArgLeu Gly Glu Val Pro Gly Ile Pro Pro Pro Asn Glu Gly Arg Phe 35 40 45 LeuGly Gly Gly Tyr Ser His Asp Asn Leu Pro Ser Asp Pro Leu Tyr 50 55 60 SerSer Ile Lys Pro Ala Leu Leu Lys Glu Ala Pro Arg Ala Glu Glu 65 70 75 80Glu Leu Pro Pro Arg Lys Val Cys Ile Val Gly Ala Gly Val Ser Gly 85 90 95Leu Tyr Ile Ala Met Ile Leu Asp Asp Leu Lys Ile Pro Asn Leu Thr 100 105110 Tyr Asp Ile Phe Glu Ser Ser Ser Arg Thr Gly Gly Arg Leu Tyr Thr 115120 125 His His Phe Thr Asp Ala Lys His Asp Tyr Tyr Asp Ile Gly Ala Met130 135 140 Arg Tyr Pro Asp Ile Pro Ser Met Lys Arg Thr Phe Asn Leu PheLys 145 150 155 160 Arg Thr Gly Met Pro Leu Ile Lys Tyr Tyr Leu Asp GlyGlu Asn Thr 165 170 175 Pro Gln Leu Tyr Asn Asn His Phe Phe Ala Lys GlyVal Ser Asp Pro 180 185 190 Tyr Met Val Ser Val Ala Asn Gly Gly Thr ValPro Asp Asp Val Val 195 200 205 Asp Ser Val Gly Glu Lys Leu Gln Gln AlaPhe Gly Tyr Tyr Lys Glu 210 215 220 Lys Leu Ala Glu Asp Phe Asp Lys GlyPhe Asp Glu Leu Met Leu Val 225 230 235 240 Asp Asp Met Thr Thr Arg GluTyr Leu Lys Arg Gly Gly Pro Lys Gly 245 250 255 Glu Ala Pro Lys Tyr AspPhe Phe Ala Ile Gln Trp Met Glu Thr Gln 260 265 270 Asn Thr Gly Thr AsnLeu Phe Asp Gln Ala Phe Ser Glu Ser Val Ile 275 280 285 Asp Ser Phe AspPhe Asp Asn Pro Thr Lys Pro Glu Trp Tyr Cys Ile 290 295 300 Glu Gly GlyThr Ser Leu Leu Val Asp Ala Met Lys Glu Thr Leu Val 305 310 315 320 HisLys Val Gln Asn Asn Lys Arg Val Asp Ala Ile Ser Ile Asp Leu 325 330 335Asp Ala Pro Asp Asp Gly Asn Met Ser Val Arg Ile Gly Gly Lys Asp 340 345350 His Ser Gly Tyr Ser Thr Val Phe Asn Thr Thr Ala Leu Gly Cys Leu 355360 365 Asp Arg Met Asp Leu Arg Gly Leu Asn Leu His Pro Thr Gln Ala Asp370 375 380 Ala Ile Arg Cys Leu His Tyr Asp Asn Ser Thr Lys Val Ala LeuLys 385 390 395 400 Phe Ser Tyr Pro Trp Trp Ile Lys Asp Cys Gly Ile ThrCys Gly Gly 405 410 415 Ala Ala Ser Thr Asp Leu Pro Leu Arg Thr Cys ValTyr Pro Ser Tyr 420 425 430 Asn Leu Asp Asp Thr Gly Glu Ala Val Leu LeuAla Ser Tyr Thr Trp 435 440 445 Ser Gln Asp Ala Thr Arg Ile Gly Ser LeuVal Lys Asp Ala Pro Pro 450 455 460 Gln Pro Pro Lys Glu Asp Glu Leu ValGlu Leu Ile Leu Gln Asn Leu 465 470 475 480 Ala Arg Leu His Ala Glu HisMet Thr Tyr Glu Lys Ile Lys Glu Ala 485 490 495 Tyr Thr Gly Val Tyr HisAla Tyr Cys Trp Ala Asn Asp Pro Asn Val 500 505 510 Gly Gly Ala Phe AlaLeu Phe Gly Pro Gly Gln Phe Ser Asn Leu Tyr 515 520 525 Pro Tyr Leu MetArg Pro Ala Ala Gly Gly Lys Phe His Ile Val Gly 530 535 540 Glu Ala SerSer Val His His Ala Trp Ile Ile Gly Ser Leu Glu Ser 545 550 555 560 AlaTyr Thr Ala Val Tyr Gln Phe Leu Tyr Lys Tyr Lys Met Trp Asp 565 570 575Tyr Leu Arg Leu Leu Leu Glu Arg Trp Gln Tyr Gly Leu Gln Glu Leu 580 585590 Glu Thr Gly Lys His Gly Thr Ala His Leu Gln Phe Ile Leu Gly Ser 595600 605 Leu Pro Lys Glu Tyr Gln Val Lys Ile 610 615 47 31 DNA ArtificialSequence Synthetic Oligonucleotide 47 cccagatcta tatttgcaaa catggacaat g31 48 33 DNA Artificial Sequence Synthetic Oligonucleotide 48 gggtctagactaacaaacat cacactttct atg 33 49 415 DNA Artificial Sequence SyntheticPolynucleotide 49 tctagaggat cagcatggcg cccaccgtga tgatggcctc gtcggccaccgccgtcgctc 60 cgttcctggg gctcaagtcc accgccagcc tccccgtcgc ccgccgctcctccagaagcc 120 tcggcaacgt cagcaacggc ggaaggatcc ggtgcatgca ggtaacaaatgcatcctagc 180 tagtagttct ttgcattgca gcagctgcag ctagcgagtt agtaataggaagggaactga 240 tgatccatgc atggactgat gtgtgttgcc catcccatcc catcccatttcccaaacgaa 300 ccgaaaacac cgtactacgt gcaggtgtgg ccctacggca acaagaagttcgagacgctg 360 tcgtacctgc cgccgctgtc gaccggcggg cgcatccgct gcatgcaggccatgg 415 50 1950 DNA Artificial Sequence Synthetic Polynucleotide 50cccgttacta ccatggctgc tatggacaat gttgactttg ctgaatctgt ccgaacccgc 60tgggcgaggc gactcattcg tgagaaggtc gccaaggaac tcaacattct aaccgaaaga 120cttggtgagg tgcccggcat ccctcctcca aatgaaggca ggttcctggg cggcggctac 180tctcacgaca atctaccgtc tgatcccctc tattccagca ttaagccggc tcttctaaag 240gaggctcctc gagcagaaga ggaactgccg cctcgaaagg tgtgcatcgt aggcgctggt 300gtttccggcc tctacatagc catgattttg gatgatttga aaatcccaaa tctcacttac 360gacatcttcg aatccagttc cagaactggt ggccgtttgt atacgcacca tttcaccgac 420gccaagcatg actattacga cattggtgct atgcgatatc ctgacatccc cagcatgaaa 480cgtaccttta acctgtttaa acgtactggg atgcctctca tcaaatatta ccttgatggc 540gagaataccc ctcagctgta caataatcac ttcttcgcca agggcgtgtc ggacccctat 600atggtgagcg tggccaatgg cggcaccgtg ccagatgatg ttgtcgatag tgttggagag 660aagttacaac aggctttcgg ttattacaaa gagaagcttg ctgaggactt cgacaaaggg 720ttcgatgagc tcatgctcgt cgacgacatg accactcgag agtacttgaa gcgaggcggg 780ccgaagggag aggcgcccaa gtatgacttt ttcgccatcc agtggatgga gacacaaaac 840actgggacaa acctgtttga tcaggccttt tctgaaagcg tcatcgactc gtttgacttt 900gacaacccga caaagcccga atggtactgc atcgagggag gaacatcgct tttggtggac 960gccatgaaag aaacccttgt ccacaaggta cagaacaaca agagagttga tgccatttcc 1020attgacttgg acgctccgga tgatgggaac atgtcggtca ggataggcgg aaaggatcac 1080tccggatata gcaccgtctt caacaccacc gctctgggct gccttgaccg catggatctg 1140cgtggtctca acttgcaccc tactcaggca gatgccattc gatgtttgca ctatgacaac 1200tcgaccaagg tggctctcaa gtttagctac ccgtggtgga tcaaggactg tggcatcact 1260tgcggtggcg cggcctcgac tgatctacct ctacgaactt gcgtttaccc atcatacaac 1320ttggacgata ctggtgaggc tgttctgctt gcctcataca cttggtctca agatgcaact 1380cgcattggat cgttggtgaa ggacgctcca ccacagccgc ccaaggagga tgagcttgtc 1440gagctgatcc tgcagaacct agcccgcctg cacgctgagc atatgaccta cgagaagatt 1500aaggaggctt acacgggcgt atatcacgcc tattgctggg ctaatgatcc caatgtcggt 1560ggtgctttcg ccctcttcgg tcccggccag ttcagcaatc tgtatccata cctgatgcgg 1620ccagcggcgg gcggcaagtt ccatatcgtc ggagaggcat ctagtgtgca tcacgcctgg 1680atcatagggt ctttggagag cgcttacacc gctgtgtacc agttcttgta caagtacaag 1740atgtgggatt acttgaggtt gttgttggag cgctggcagt atggtctcca ggagttagag 1800acggggaagc acggtacggc tcatttgcag tttattctag gttcacttcc caaggagtac 1860caggtgaaga tttaaagcga aagaggtact acggcatgga gacaattttg ggtagagatt 1920ctagtattcc agcagtttca taataataag 1950 51 34 DNA Artificial SequenceSynthetic Oligonucleotide 51 cccgttacta ccatggctgc tatggacaat gttg 34 5234 DNA Artificial Sequence Synthetic Oligonucleotide 52 ctaacaaacagaattcttat tattatgaaa ctgc 34 53 193 DNA Artificial Sequence SyntheticPolynucleotide 53 tctagaccgg gcatggcgtc ccgccgggtc ttctcgtcca tccttcgctctgcctctcgc 60 attcgctccg cctcaccgtc cccatgcccg cgtgcgccgc tccaccaccgcccgtccccc 120 gcgggcttca tactcaaccg tgtagccgcc tacgcctcct ccgccacggcccaggcggca 180 cctgccatgg cgc 193 54 34 PRT Artificial SequenceSynthetic Polypeptide 54 Met Glu Asp Ala Ala Ala Arg Arg Met Arg Leu AlaSer His Leu Arg 1 5 10 15 Pro Pro Ala Ser Gln Met Glu Glu Ser Pro LeuLeu Arg Gly Ser Asn 20 25 30 Cys Arg 55 107 DNA Artificial SequenceSynthetic Oligonucleotide 55 ccatggagga cgcagcagca aggcggatgg agaggctcgcctcccacctc cgcccgcccg 60 cttctcagat ggaggaatca cccctcctga ggggctccaattgccgg 107 56 617 PRT Artificial Sequence Synthetic Polypeptide 56 MetAsp Asn Val Asp Phe Ala Glu Ser Val Arg Thr Arg Trp Ala Arg 1 5 10 15Arg Leu Ile Arg Glu Lys Val Ala Lys Glu Leu Asn Ile Leu Thr Glu 20 25 30Arg Leu Gly Glu Val Pro Gly Ile Pro Pro Pro Asn Glu Gly Arg Phe 35 40 45Leu Gly Gly Gly Tyr Ser His Asp Asn Leu Pro Ser Asp Pro Leu Tyr 50 55 60Ser Ser Ile Lys Pro Ala Leu Leu Gly Gly Gly Gly Xaa Xaa Xaa Xaa 65 70 7580 Xaa Xaa Gly Gly Gly Gly Val Cys Ile Val Gly Ala Gly Val Ser Gly 85 9095 Leu Tyr Ile Ala Met Ile Leu Asp Asp Leu Lys Ile Pro Asn Leu Thr 100105 110 Tyr Asp Ile Phe Glu Ser Ser Ser Arg Thr Gly Gly Arg Leu Tyr Thr115 120 125 His His Phe Thr Asp Ala Lys His Asp Tyr Tyr Asp Ile Gly AlaMet 130 135 140 Arg Tyr Pro Asp Ile Pro Ser Met Lys Arg Thr Phe Asn LeuPhe Lys 145 150 155 160 Arg Thr Gly Met Pro Leu Ile Lys Tyr Tyr Leu AspGly Glu Asn Thr 165 170 175 Pro Gln Leu Tyr Asn Asn His Phe Phe Ala LysGly Val Ser Asp Pro 180 185 190 Tyr Met Val Ser Val Ala Asn Gly Gly ThrVal Pro Asp Asp Val Val 195 200 205 Asp Ser Val Gly Glu Lys Leu Gln GlnAla Phe Gly Tyr Tyr Lys Glu 210 215 220 Lys Leu Ala Glu Asp Phe Asp LysGly Phe Asp Glu Leu Met Leu Val 225 230 235 240 Asp Asp Met Thr Thr ArgGlu Tyr Leu Lys Arg Gly Gly Pro Lys Gly 245 250 255 Glu Ala Pro Lys TyrAsp Phe Phe Ala Ile Gln Trp Met Glu Thr Gln 260 265 270 Asn Thr Gly ThrAsn Leu Phe Asp Gln Ala Phe Ser Glu Ser Val Ile 275 280 285 Asp Ser PheAsp Phe Asp Asn Pro Thr Lys Pro Glu Trp Tyr Cys Ile 290 295 300 Glu GlyGly Thr Ser Leu Leu Val Asp Ala Met Lys Glu Thr Leu Val 305 310 315 320His Lys Val Gln Asn Asn Lys Arg Val Asp Ala Ile Ser Ile Asp Leu 325 330335 Asp Ala Pro Asp Asp Gly Asn Met Ser Val Arg Ile Gly Gly Lys Asp 340345 350 His Ser Gly Tyr Ser Thr Val Phe Asn Thr Thr Ala Leu Gly Cys Leu355 360 365 Asp Arg Met Asp Leu Arg Gly Leu Asn Leu His Pro Thr Gln AlaAsp 370 375 380 Ala Ile Arg Cys Leu His Tyr Asp Asn Ser Thr Lys Val AlaLeu Lys 385 390 395 400 Phe Ser Tyr Pro Trp Trp Ile Lys Asp Cys Gly IleThr Cys Gly Gly 405 410 415 Ala Ala Ser Thr Asp Leu Pro Leu Arg Thr CysVal Tyr Pro Ser Tyr 420 425 430 Asn Leu Asp Asp Thr Gly Glu Ala Val LeuLeu Ala Ser Tyr Thr Trp 435 440 445 Ser Gln Asp Ala Thr Arg Ile Gly SerLeu Val Lys Asp Ala Pro Pro 450 455 460 Gln Pro Pro Lys Glu Asp Glu LeuVal Glu Leu Ile Leu Gln Asn Leu 465 470 475 480 Ala Arg Leu His Ala GluHis Met Thr Tyr Glu Lys Ile Lys Glu Ala 485 490 495 Tyr Thr Gly Val TyrHis Ala Tyr Cys Trp Ala Asn Asp Pro Asn Val 500 505 510 Gly Gly Ala PheAla Leu Phe Gly Pro Gly Gln Phe Ser Asn Leu Tyr 515 520 525 Pro Tyr LeuMet Arg Pro Ala Ala Gly Gly Lys Phe His Ile Val Gly 530 535 540 Glu AlaSer Ser Val His His Ala Trp Ile Ile Gly Ser Leu Glu Ser 545 550 555 560Ala Tyr Thr Ala Val Tyr Gln Phe Leu Tyr Lys Tyr Lys Met Trp Asp 565 570575 Tyr Leu Arg Leu Leu Leu Glu Arg Trp Gln Tyr Gly Leu Gln Glu Leu 580585 590 Glu Thr Gly Lys His Gly Thr Ala His Leu Gln Phe Ile Leu Gly Ser595 600 605 Leu Pro Lys Glu Tyr Gln Val Lys Ile 610 615 57 617 PRTArtificial Sequence Synthetic Polypeptide 57 Met Asp Asn Val Asp Phe AlaGlu Ser Val Arg Thr Arg Trp Ala Arg 1 5 10 15 Arg Leu Ile Arg Glu LysVal Ala Lys Glu Leu Asn Ile Leu Thr Glu 20 25 30 Arg Leu Gly Glu Val ProGly Ile Pro Pro Pro Asn Glu Gly Arg Phe 35 40 45 Leu Gly Gly Gly Tyr SerHis Asp Asn Leu Pro Ser Asp Pro Leu Tyr 50 55 60 Ser Ser Ile Gly Gly GlySer Gly Gly Xaa Xaa Xaa Xaa Xaa Xaa Gly 65 70 75 80 Gly Gly Pro Pro ArgLys Val Cys Ile Val Gly Ala Gly Val Ser Gly 85 90 95 Leu Tyr Ile Ala MetIle Leu Asp Asp Leu Lys Ile Pro Asn Leu Thr 100 105 110 Tyr Asp Ile PheGlu Ser Ser Ser Arg Thr Gly Gly Arg Leu Tyr Thr 115 120 125 His His PheThr Asp Ala Lys His Asp Tyr Tyr Asp Ile Gly Ala Met 130 135 140 Arg TyrPro Asp Ile Pro Ser Met Lys Arg Thr Phe Asn Leu Phe Lys 145 150 155 160Arg Thr Gly Met Pro Leu Ile Lys Tyr Tyr Leu Asp Gly Glu Asn Thr 165 170175 Pro Gln Leu Tyr Asn Asn His Phe Phe Ala Lys Gly Val Ser Asp Pro 180185 190 Tyr Met Val Ser Val Ala Asn Gly Gly Thr Val Pro Asp Asp Val Val195 200 205 Asp Ser Val Gly Glu Lys Leu Gln Gln Ala Phe Gly Tyr Tyr LysGlu 210 215 220 Lys Leu Ala Glu Asp Phe Asp Lys Gly Phe Asp Glu Leu MetLeu Val 225 230 235 240 Asp Asp Met Thr Thr Arg Glu Tyr Leu Lys Arg GlyGly Pro Lys Gly 245 250 255 Glu Ala Pro Lys Tyr Asp Phe Phe Ala Ile GlnTrp Met Glu Thr Gln 260 265 270 Asn Thr Gly Thr Asn Leu Phe Asp Gln AlaPhe Ser Glu Ser Val Ile 275 280 285 Asp Ser Phe Asp Phe Asp Asn Pro ThrLys Pro Glu Trp Tyr Cys Ile 290 295 300 Glu Gly Gly Thr Ser Leu Leu ValAsp Ala Met Lys Glu Thr Leu Val 305 310 315 320 His Lys Val Gln Asn AsnLys Arg Val Asp Ala Ile Ser Ile Asp Leu 325 330 335 Asp Ala Pro Asp AspGly Asn Met Ser Val Arg Ile Gly Gly Lys Asp 340 345 350 His Ser Gly TyrSer Thr Val Phe Asn Thr Thr Ala Leu Gly Cys Leu 355 360 365 Asp Arg MetAsp Leu Arg Gly Leu Asn Leu His Pro Thr Gln Ala Asp 370 375 380 Ala IleArg Cys Leu His Tyr Asp Asn Ser Thr Lys Val Ala Leu Lys 385 390 395 400Phe Ser Tyr Pro Trp Trp Ile Lys Asp Cys Gly Ile Thr Cys Gly Gly 405 410415 Ala Ala Ser Thr Asp Leu Pro Leu Arg Thr Cys Val Tyr Pro Ser Tyr 420425 430 Asn Leu Asp Asp Thr Gly Glu Ala Val Leu Leu Ala Ser Tyr Thr Trp435 440 445 Ser Gln Asp Ala Thr Arg Ile Gly Ser Leu Val Lys Asp Ala ProPro 450 455 460 Gln Pro Pro Lys Glu Asp Glu Leu Val Glu Leu Ile Leu GlnAsn Leu 465 470 475 480 Ala Arg Leu His Ala Glu His Met Thr Tyr Glu LysIle Lys Glu Ala 485 490 495 Tyr Thr Gly Val Tyr His Ala Tyr Cys Trp AlaAsn Asp Pro Asn Val 500 505 510 Gly Gly Ala Phe Ala Leu Phe Gly Pro GlyGln Phe Ser Asn Leu Tyr 515 520 525 Pro Tyr Leu Met Arg Pro Ala Ala GlyGly Lys Phe His Ile Val Gly 530 535 540 Glu Ala Ser Ser Val His His AlaTrp Ile Ile Gly Ser Leu Glu Ser 545 550 555 560 Ala Tyr Thr Ala Val TyrGln Phe Leu Tyr Lys Tyr Lys Met Trp Asp 565 570 575 Tyr Leu Arg Leu LeuLeu Glu Arg Trp Gln Tyr Gly Leu Gln Glu Leu 580 585 590 Glu Thr Gly LysHis Gly Thr Ala His Leu Gln Phe Ile Leu Gly Ser 595 600 605 Leu Pro LysGlu Tyr Gln Val Lys Ile 610 615 58 617 PRT Artificial Sequence SyntheticPolypeptide 58 Met Asp Asn Val Asp Phe Ala Glu Ser Val Arg Thr Arg TrpAla Arg 1 5 10 15 Arg Leu Ile Arg Glu Lys Val Ala Lys Glu Leu Asn IleLeu Thr Glu 20 25 30 Arg Leu Gly Glu Val Pro Gly Ile Pro Pro Pro Asn GluGly Arg Phe 35 40 45 Leu Gly Gly Gly Tyr Ser His Asp Asn Leu Pro Ser AspPro Leu Tyr 50 55 60 Ser Ser Ile Lys Pro Gly Gly Gly Gly Xaa Xaa Xaa XaaXaa Xaa Gly 65 70 75 80 Gly Gly Pro Pro Arg Lys Val Cys Ile Val Gly AlaGly Val Ser Gly 85 90 95 Leu Tyr Ile Ala Met Ile Leu Asp Asp Leu Lys IlePro Asn Leu Thr 100 105 110 Tyr Asp Ile Phe Glu Ser Ser Ser Arg Thr GlyGly Arg Leu Tyr Thr 115 120 125 His His Phe Thr Asp Ala Lys His Asp TyrTyr Asp Ile Gly Ala Met 130 135 140 Arg Tyr Pro Asp Ile Pro Ser Met LysArg Thr Phe Asn Leu Phe Lys 145 150 155 160 Arg Thr Gly Met Pro Leu IleLys Tyr Tyr Leu Asp Gly Glu Asn Thr 165 170 175 Pro Gln Leu Tyr Asn AsnHis Phe Phe Ala Lys Gly Val Ser Asp Pro 180 185 190 Tyr Met Val Ser ValAla Asn Gly Gly Thr Val Pro Asp Asp Val Val 195 200 205 Asp Ser Val GlyGlu Lys Leu Gln Gln Ala Phe Gly Tyr Tyr Lys Glu 210 215 220 Lys Leu AlaGlu Asp Phe Asp Lys Gly Phe Asp Glu Leu Met Leu Val 225 230 235 240 AspAsp Met Thr Thr Arg Glu Tyr Leu Lys Arg Gly Gly Pro Lys Gly 245 250 255Glu Ala Pro Lys Tyr Asp Phe Phe Ala Ile Gln Trp Met Glu Thr Gln 260 265270 Asn Thr Gly Thr Asn Leu Phe Asp Gln Ala Phe Ser Glu Ser Val Ile 275280 285 Asp Ser Phe Asp Phe Asp Asn Pro Thr Lys Pro Glu Trp Tyr Cys Ile290 295 300 Glu Gly Gly Thr Ser Leu Leu Val Asp Ala Met Lys Glu Thr LeuVal 305 310 315 320 His Lys Val Gln Asn Asn Lys Arg Val Asp Ala Ile SerIle Asp Leu 325 330 335 Asp Ala Pro Asp Asp Gly Asn Met Ser Val Arg IleGly Gly Lys Asp 340 345 350 His Ser Gly Tyr Ser Thr Val Phe Asn Thr ThrAla Leu Gly Cys Leu 355 360 365 Asp Arg Met Asp Leu Arg Gly Leu Asn LeuHis Pro Thr Gln Ala Asp 370 375 380 Ala Ile Arg Cys Leu His Tyr Asp AsnSer Thr Lys Val Ala Leu Lys 385 390 395 400 Phe Ser Tyr Pro Trp Trp IleLys Asp Cys Gly Ile Thr Cys Gly Gly 405 410 415 Ala Ala Ser Thr Asp LeuPro Leu Arg Thr Cys Val Tyr Pro Ser Tyr 420 425 430 Asn Leu Asp Asp ThrGly Glu Ala Val Leu Leu Ala Ser Tyr Thr Trp 435 440 445 Ser Gln Asp AlaThr Arg Ile Gly Ser Leu Val Lys Asp Ala Pro Pro 450 455 460 Gln Pro ProLys Glu Asp Glu Leu Val Glu Leu Ile Leu Gln Asn Leu 465 470 475 480 AlaArg Leu His Ala Glu His Met Thr Tyr Glu Lys Ile Lys Glu Ala 485 490 495Tyr Thr Gly Val Tyr His Ala Tyr Cys Trp Ala Asn Asp Pro Asn Val 500 505510 Gly Gly Ala Phe Ala Leu Phe Gly Pro Gly Gln Phe Ser Asn Leu Tyr 515520 525 Pro Tyr Leu Met Arg Pro Ala Ala Gly Gly Lys Phe His Ile Val Gly530 535 540 Glu Ala Ser Ser Val His His Ala Trp Ile Ile Gly Ser Leu GluSer 545 550 555 560 Ala Tyr Thr Ala Val Tyr Gln Phe Leu Tyr Lys Tyr LysMet Trp Asp 565 570 575 Tyr Leu Arg Leu Leu Leu Glu Arg Trp Gln Tyr GlyLeu Gln Glu Leu 580 585 590 Glu Thr Gly Lys His Gly Thr Ala His Leu GlnPhe Ile Leu Gly Ser 595 600 605 Leu Pro Lys Glu Tyr Gln Val Lys Ile 610615

What is claimed is:
 1. A composition for controlling insect infestationof plants comprising a mixture of: a first enzyme comprising an aminoacid oxidase; and a second enzyme that provides insecticidal activitywhen present in said mixture with said first enzyme; wherein saidmixture is ingested by an insect.
 2. The composition of claim 1, whereinsaid first enzyme is a lysine oxidase and said second enzyme convertsΔ1-piperideine-2-carboxylate to tedanalactam.
 3. The composition ofclaim 2, wherein said second enzyme is approximately M_(r) 50,000. 4.The composition of claim 3, wherein: said second enzyme is isolated froman extract of a fungal species fermentation; and said extract exhibitscoleopteran insecticidal activity.
 5. The composition of claim 4,wherein the fungal species is a Trichoderma.
 6. The composition of claim4, wherein said insecticidal activity is independent of tedanalactamsynthase activity.
 7. The composition of claim 4, wherein saidcoleopteran insecticidal activity is effective in controllingcoleopteran species selected from the group consisting of Diabrotica,Melanotus, Leptinotarsa, and Anthonomus.
 8. The composition of claim 7,wherein said coleopteran insecticidal activity is effective incontrolling insects selected from the group consisting of boll weevil(BWV), corn rootworm (CRW), wireworm (WW), and Colorado potato beetle(CPB).
 9. The composition of claim 1, wherein said first enzyme and saidsecond enzyme are present respectively in said mixture in a molar ratioof about 100:1 to about 1:1.
 10. The composition of claim 1, whereinsaid first enzyme and said second enzyme are present respectively insaid mixture in a molar ratio of about 10:1 to about 1:1.
 11. Thecomposition of claim 1, wherein said second enzyme and said first enzymeare present respectively in said mixture in a ratio of about 100:1 toabout 1:1.
 12. The composition of claim 1, wherein said second enzymeand said first enzyme are present respectively in said mixture in aratio of about 10:1 to about 1:1.
 13. A method of controlling insectinfestation of plants comprising providing a combination of: a firstenzyme comprising an amino acid oxidase; and a second enzyme thatprovides insecticidal activity when present in a mixture with said firstenzyme; wherein said mixture is ingested by an insect.
 14. The method ofclaim 13, wherein said first enzyme is a lysine oxidase and said secondenzyme converts Δ1-piperideine-2-carboxylate to tedanalactam.
 15. Themethod of claim 14, wherein said second enzyme is approximately M_(r)50,000.
 16. The method of claim 15, wherein: said second enzyme isisolated from an extract of a fungal species fermentation; and saidextract exhibits coleopteran insecticidal activity.
 17. The method ofclaim 16, wherein the fungal species is a Trichoderma.
 18. The method ofclaim 16, wherein said insecticidal activity is independent oftedanalactam synthase activity.
 19. The method of claim 16, wherein saidcoleopteran insecticidal activity is effective in controllingcoleopteran species selected from the group consisting of Diabrotica,Melanotus, Leptinotarsa, and Anthonomus.
 20. The method of claim 19,wherein said coleopteran insecticidal activity is effective incontrolling insects selected from the group consisting of boll weevil(BWV), corn rootworm (CRW), wireworm (WW), and Colorado potato beetle(CPB).
 21. The method of claim 13, wherein said first enzyme and saidsecond enzyme are present respectively in said mixture in a molar ratioof about 100:1 to about 1:1.
 22. The method of claim 13, wherein saidfirst enzyme and said second enzyme are present respectively in saidmixture in a molar ratio of about 10:1 to about 1:1.
 23. The method ofclaim 13, wherein said second enzyme and said first enzyme are presentrespectively in said mixture in a ratio of about 100:1 to about 1:1. 24.The method of claim 13, wherein said second enzyme and said first enzymeare present respectively in said mixture in a ratio of about 10:1 toabout 1:1.
 25. A method of controlling insect infestation of plantscomprising providing a plant containing a composition comprising: afirst enzyme comprising an amino acid oxidase; and a second enzyme thatprovides insecticidal activity when present in a mixture with said firstenzyme; wherein said plant is ingested by an insect.
 26. The method ofclaim 25 wherein said first enzyme is a lysine oxidase and said secondenzyme converts Δ1-piperideine-2-carboxylate to tedanalactam.
 27. Themethod of claim 26, wherein said second enzyme is approximately M_(r)50,000.
 28. The method of claim 27, wherein: said second enzyme isisolated from an extract of a fungal species fermentation; and saidextract exhibits coleopteran insecticidal activity.
 29. The method ofclaim 28, wherein said fungal species is a Trichoderma.
 30. The methodof claim 28, wherein said insecticidal activity is independent oftedanalactam synthase activity.
 31. The method of claim 28, wherein saidcoleopteran insecticidal activity is effective in controllingcoleopteran species selected from the group consisting of Diabrotica,Melanotus, Leptinotarsa, and Anthonomus.
 32. The method of claim 31,wherein said coleopteran insecticidal activity is effective incontrolling insects selected from the group consisting of boll weevil(BWV), corn rootworm (CRW), wireworm (WW), and Colorado potato beetle(CPB).
 33. The method of claim 25, wherein said first enzyme and saidsecond enzyme are present respectively in said mixture in a molar ratioof about 100:1 to about 1:1.
 34. The method of claim 25, wherein saidfirst enzyme and said second enzyme are present respectively in saidmixture in a molar ratio of about 10:1 to about 1:1.
 35. The method ofclaim 25, wherein said second enzyme and said first enzyme are presentrespectively in said mixture in a molar ratio of about 100:1 to about1:1.
 36. The method of claim 25, wherein said second enzyme and saidfirst enzyme are present respectively in said mixture in a molar ratioof about 10:1 to about 1:1.
 37. A method of controlling insectinfestation of plants comprising providing a plurality of plant cellscontaining a composition comprising: a first enzyme comprising an aminoacid oxidase; and a second enzyme that provides insecticidal activitywhen present in a mixture with said first enzyme; wherein said plantcells are ingested by an insect.
 38. The method of claim 37, whereinsaid first enzyme is a lysine oxidase and said second enzyme convertsΔ1-piperideine-2-carboxylate to tedanalactam.
 39. The method of claim38, wherein said second enzyme is approximately M_(r) 50,000.
 40. Themethod of claim 37, wherein: said second enzyme is isolated from anextract of a fungal species fermentation; and said extract exhibitscoleopteran insecticidal activity.
 41. The method of claim 40, whereinthe fungal species is a Trichoderma.
 42. The method of claim 40, whereinsaid insecticidal activity is independent of tedanalactam synthaseactivity.
 43. The method of claim 40, wherein said coleopteraninsecticidal activity is effective in controlling coleopteran speciesselected from the group consisting of Diabrotica, Melanotus,Leptinotarsa, and Anthonomus.
 44. The method of claim 43, wherein saidcoleopteran insecticidal activity is effective in controlling insectsselected from the group consisting of boll weevil (BWV), corn rootworm(CRW), wireworm (WW), and Colorado potato beetle (CPB).
 45. The methodof claim 37, wherein said first enzyme and said second enzyme arepresent respectively in said mixture in a molar ratio of about 100:1 toabout 1:1.
 46. The method of claim 37, wherein said first enzyme andsaid second enzyme are present respectively in said mixture in a molarratio of about 10:1 to about 1:1.
 47. The method of claim 37, whereinsaid second enzyme and said first enzyme are present respectively insaid mixture in a molar ratio of about 100:1 to about 1:1.
 48. Themethod of claim 37, wherein said second enzyme and said first enzyme arepresent respectively in said mixture in a molar ratio of about 1 0:1 toabout 1:1.
 49. A method of controlling insect infestation of plantscomprising providing a plurality of bacterial cells containing acomposition comprising: a first enzyme comprising an amino acid oxidase;and a second enzyme that provides insecticidal activity when present ina mixture with said first enzyme; wherein said bacterial cells areingested by an insect.
 50. The method of claim 49 wherein said firstenzyme is a lysine oxidase and said second enzyme convertsΔ1-piperideine-2-carboxylate to tedanalactam.
 51. The method of claim50, wherein said second enzyme is approximately M_(r) 50,000.
 52. Themethod of claim 49, wherein: said second enzyme is isolated from anextract of a fungal species fermentation; and said extract exhibitscoleopteran insecticidal activity.
 53. The method of claim 52, whereinthe fungal species is a Trichoderma.
 54. The method of claim 52, whereinsaid insecticidal activity is independent of tedanalactam synthaseactivity.
 55. The method of claim 52, wherein said coleopteraninsecticidal activity is effective in controlling coleopteran speciesselected from the group consisting of Diabrotica, Melanotus,Leptinotarsa, and Anthonomus.
 56. The method of claim 55, wherein saidcoleopteran insecticidal activity is effective in controlling insectsselected from the group consisting of boll weevil (BVWV), corn rootworm(CRW), wireworm (WW), and Colorado potato beetle (CPB).
 57. The methodof claim 49, wherein said first enzyme and said second enzyme arepresent respectively in said mixture in a molar ratio of about 100:1 toabout 1:1.
 58. The method of claim 49, wherein said first enzyme andsaid second enzyme are present respectively in said mixture in a molarratio of about 10:1 to about 1:1.
 59. The method of claim 49, whereinsaid second enzyme and said first enzyme are present respectively insaid mixture in a molar ratio of about 100:1 to about 1:1.
 60. Themethod of claim 49, wherein said second enzyme and said first enzyme arepresent respectively in said mixture in a molar ratio of about 10:1 toabout 1:1.
 61. A structural gene encoding a lysine oxidase enzyme orproenzyme, said structural gene selected from the group consisting of:a) a DNA sequence at least 80% identical to that indicated in SEQ ID NO:15 from base number 663 to base number 2513; b) a DNA sequence encodingan amino acid sequence at least 80% identical to that indicated in SEQID NO: 46; and c) a DNA sequence that hybridizes under stringentconditions with the DNA sequence indicated in SEQ ID NO: 15 from basenumber 663 to base number 2513, or the complement thereof.
 62. Thestructural gene of claim 61 having the DNA sequence of SEQ ID NO: 15from base number 663 to base number
 2513. 63. A structural gene encodinga lysine oxidase protein, said protein comprising a variant of a lysineoxidase or proenzyme susceptible to cleavage and enzymatic activation byinsect midgut proteases but resistant to cleavage and enzymaticactivation by endogenous plant proteases.
 64. A structural gene encodingan enzyme that provides insecticidal activity when combined with anamino acid oxidase, said structural gene comprising a gene selected fromthe group consisting of: a) a DNA sequence at least 80% identical tothat indicated in SEQ ID NO: 40 from base number 12 to base number 1349;b) a DNA sequence encoding an amino acid sequence at least 80% identicalto the amino acid sequence indicated in SEQ ID NO: 41; and c) a DNAsequence that hybridizes under stringent conditions with the DNAsequence indicated in SEQ ID NO: 40 from base number 12 to base number1349, or to the complement thereof.
 65. The structural gene of claim 64,wherein the amino acid oxidase is a lysine oxidase or lysine oxidaseproenzyme.
 66. A structural gene of claim 64, wherein said enzymeconverts Δ1-piperideine-2-carboxylate to tedanalactam.
 67. Thestructural gene of claim 66 having the DNA sequence of SEQ ID NO: 40from base number 12 to base number
 1349. 68. The structural gene ofclaim 66, wherein said enzyme is approximately M_(r) 50,000.
 69. Thestructural gene of claim 64, wherein: said enzyme is isolated from anextract of a fungal species fermentation; and said extract exhibitscoleopteran insecticidal activity.
 70. The structural gene of claim 69,wherein the fungal species is a Trichoderma.
 71. The structural gene ofclaim 69, wherein said insecticidal activity is independent oftedanalactam synthase activity.
 72. The structural gene of claim 69,wherein said coleopteran insecticidal activity is effective incontrolling coleopteran species selected from the group consisting ofDiabrotica, Melanotus, Leptinotarsa, and Anthonomus.
 73. The structuralgene of claim 72, wherein said coleopteran insecticidal activity iseffective in controlling insects selected from the group consisting ofboll weevil (BWV), corn rootworm (CRW), wireworm (WW), and Coloradopotato beetle (CPB).
 74. An enzyme that provides insecticidal activitywhen present in a mixture with an amino acid oxidase, wherein saidenzyme is isolated from an extract of a fungal species fermentation andis substantially free of other proteinaceous materials.
 75. The enzymeof claim 74, wherein said enzyme converts Δ1-piperideine-2-carboxylateto tedanalactam.
 76. The enzyme of claim 75, wherein said enzyme isapproximately M_(r) 50,000.
 77. The enzyme of claim 74, wherein thefungal species is a Trichoderma.
 78. The enzyme of claim 74, whereinsaid insecticidal activity is independent of tedanalactam synthaseactivity.
 79. The enzyme of claim 74, wherein said coleopteraninsecticidal activity is effective in controlling coleopteran speciesselected from the group consisting of Diabrotica, Melanotus,Leptinotarsa, and Anthonomus.
 80. The enzyme of claim 79, wherein saidcoleopteran insecticidal activity is effective in controlling insectsselected from the group consisting of boll weevil (BWV), corn rootworm(CRW), wireworm (WW), and Colorado potato beetle (CPB).
 81. A pluralityof genetically transformed plant cells expressing one or more geneswhich encode a composition comprising: a first enzyme comprising anamino acid oxidase enzyme; and a second enzyme that providesinsecticidal activity when present in a mixture with said first enzyme;wherein said composition is effective in controlling coleopteran insectsthat ingest said genetically transformed plant cells.
 82. The pluralityof genetically transformed plant cells of claim 81, wherein said aminoacid oxidase enzyme is a lysine oxidase enzyme and said second enzymeconverts Δ1-piperideine-2-carboxylate to tedanalactam.
 83. The pluralityof genetically transformed plant cells of claim 82, wherein said secondenzyme is approximately M_(r) 50,000.
 84. The plurality of geneticallytransformed cells of claim 81, wherein: said second enzyme is isolatedfrom an extract of a fungal species fermentation; and said extractexhibits coleopteran insecticidal activity.
 85. The plurality ofgenetically transformed plant cells of claim 84, wherein the fungalspecies is a Trichoderma.
 86. The plurality of genetically transformedplant cells of claim 84, wherein said insecticidal activity isindependent of tedanalactam synthase activity.
 87. The plurality ofgenetically transformed plant cells of claim 84, wherein saidcoleopteran insecticidal activity is effective in controllingcoleopteran species selected from the group consisting of Diabrotica,Melanotus, Leptinotarsa, and Anthonomus.
 88. The plurality ofgenetically transformed plant cells of claim 87, wherein saidcoleopteran insecticidal activity is effective in controlling insectsselected from the group consisting of boll weevil (BWV), corn rootworm(CRW), wireworm (WW), and Colorado potato beetle (CPB).
 89. Agenetically transformed plant expressing one or more genes which encodea composition comprising: a first enzyme comprising an amino acidoxidase enzyme; and a second enzyme that provides insecticidal activitywhen present in a mixture with said first enzyme; wherein saidcomposition is effective in controlling coleopteran insects that ingestsaid genetically transformed plant.
 90. The genetically transformedplant of claim 89, wherein said amino acid oxidase enzyme is a lysineoxidase enzyme and said second enzyme convertsΔ1-piperideine-2-carboxylate to tedanalactam.
 91. The geneticallytransformed plant of claim 90, wherein said second enzyme isapproximately M_(r) 50,000.
 92. The genetically transformed plant ofclaim 89, wherein: said second enzyme is isolated from an extract of afungal species fermentation; and said extract exhibits coleopteraninsecticidal activity.
 93. The genetically transformed plant of claim92, wherein said fungal species is a Trichoderma.
 94. The geneticallytransformed plant of claim 92, wherein said insecticidal activity isindependent of tedanalactam synthase activity.
 95. The geneticallytransformed plant of claim 92, wherein said coleopteran insecticidalactivity is effective in controlling coleopteran species selected fromthe group consisting of Diabrotica, Melanotus, Leptinotarsa, andAnthonomus.
 96. The genetically transformed plant of claim 95, whereinsaid coleopteran insecticidal activity is effective in controllinginsects selected from the group consisting of boll weevil (BWV), cornrootworm (CRW), wireworm (WW), and Colorado potato beetle (CPB).
 97. Thegenetically transformed plant of claim 89, wherein said plant isselected from the group of genera consisting of Fabaceae, Medicago,Trifolium, Vigna, Daucus, Arabidopsis, Brassica, Raphanus, Sinapis,Lycopersicon, Capsicum, Solanum, Nicotiana, Helianthus, Bromus,Asparagus, Panicum, Pennisetum, Cucumis, Lolium, Glycine,Passifloraceae, Triticum, Gossypium, and Zea.
 98. A plurality ofgenetically transformed bacterial cells expressing one or more geneswhich encode a composition comprising: a) a first enzyme comprising anamino acid oxidase enzyme; and b) a second enzyme that providesinsecticidal activity when present in mixture with said first enzyme;wherein said composition is effective in controlling coleopteran insectsthat ingest said genetically transformed bacterial cells.
 99. Thebacterial cells of claim 98, wherein said amino acid oxidase enzyme is alysine oxidase enzyme and said second enzyme convertsΔ1-piperideine-2-carboxylate to tedanalactam.
 100. The bacterial cellsof claim 99, wherein said second enzyme is approximately M_(r) 50,000.101. The bacterial cells of claim 98, wherein: said second enzyme isisolated from an extract of a fungal species fermentation; and saidextract exhibits coleopteran insecticidal activity.
 102. The bacterialcells of claim 101, wherein the fungal species is a Trichoderma. 103.The bacterial cells of claim 101, wherein said insecticidal activity isindependent of tedanalactam synthase activity.
 104. The bacterial cellsof claim 101, wherein said coleopteran insecticidal activity iseffective in controlling coleopteran species selected from the groupconsisting of Diabrotica, Melanotus, Leptinotarsa, and Anthonomus. 105.The bacterial cells of claim 104, wherein said coleopteran insecticidalactivity is effective in controlling insects selected from the groupconsisting of boll weevil (BWV), corn rootworm (CRW), wireworm (WW), andColorado potato beetle (CPB).
 106. The bacterial cells of claim 98,wherein said bacterial cells are of the species Pseudomonas.
 107. Thebacterial cells of claim 98, wherein said bacterial cells are of thespecies Agrobacterium.
 108. The bacterial cells of claim 98, whereinsaid bacterial cells are of the species Clavibacter.
 109. A geneticallytransformed plant containing a double stranded DNA molecule, whereinsaid DNA molecule comprises: a) a promoter sequence which functions inplants to cause the production of an RNA sequence, operably linked to;b) a first structural gene encoding a first enzyme comprising an aminoacid oxidase enzyme; c) a second structural gene encoding a secondenzyme that provides insecticidal activity when present in a mixturewith said first enzyme; and d) a 3′ non-translated DNA sequence whichfunctions in plants to cause the addition of a polyadenylated nucleotidesequence to the 3′ end of said RNA sequence; wherein said DNA moleculeencodes an in frame translational fusion protein of said amino acidoxidase enzyme and said second enzyme.
 110. The genetically transformedplant of claim 109, wherein said amino acid oxidase enzyme is a lysineoxidase enzyme and said second enzyme convertsΔ1-piperideine-2-carboxylate to tedanalactam.
 111. The geneticallytransformed plant of claim 110, wherein said second enzyme isapproximately M_(r) 50,000.
 112. The genetically transformed plant ofclaim 109, wherein: said second enzyme is isolated from an extract of afungal species fermentation; and said extract exhibits coleopteraninsecticidal activity.
 113. The genetically transformed plant of claim112, wherein the fungal species is a Trichoderma.
 114. The geneticallytransformed plant of claim 113, wherein said insecticidal activity isindependent of tedanalactam synthase activity.
 115. The geneticallytransformed plant of claim 112, wherein said coleopteran insecticidalactivity is effective in controlling coleopteran species selected fromthe group consisting of Diabrotica, Melanotus, Leptinotarsa, andAnthonomus.
 116. The genetically transformed plant of claim 115, whereinsaid coleopteran insecticidal activity is effective in controllinginsects selected from the group consisting of boll weevil (BWV), cornrootworm (CRW), wireworm (WW), and Colorado potato beetle (CPB). 117.The genetically transformed plant of claim 109, wherein a plantendogenous endoprotease is capable of post-translationally cleaving saidfusion protein to produce an insecticidally active compositioncomprising said first enzyme and said second enzyme.
 118. Thegenetically transformed plant of claim 109, wherein an insect endogenousendoprotease is capable of post-translationally cleaving said fusionprotein to produce an insecticidally active composition comprising saidfirst enzyme and said second enzyme upon ingestion of said plant by aninsect.
 119. The genetically transformed plant of claim 109, whereinsaid promoter sequence is selected from the group of root tissueenhanced or root tissue specific promoter sequences consisting of theCaMV derived AS4 promoter sequence, tobacco RB7 promoter sequence, andthe rice RC2 promoter sequence.
 120. The genetically transformed plantof claim 109, wherein said promoter sequence is selected from the groupof plant promoter sequences consisting of nopaline synthase promotersequence (NOS), octopine synthase promoter sequence (OCS), cauliflowermosaic virus 19S promoter sequence (CaMV19S), cauliflower mosaic virus35S promoter sequence (CaMV35S), ribulose 1,5-bisphosphate carboxylasesmall subunit promoter sequence (ssRUBISCO), and the figwort mosaicvirus (FMV) promoter sequence.
 121. A DNA vector comprising: a) apromoter which functions in a plant cell to cause the production of anRNA sequence, operably linked to a polynucleotide cassette comprising;b) a 5′ non-translated leader sequence; c) a structural gene encoding anenzyme capable of converting Δ1-piperideine-2-carboxylate totedanalactam; and d) a DNA sequence which functions in plants to causethe addition of a 3′ non-translated polyadenylated nucleotide sequenceto the 3′ end of said RNA sequence; wherein expression of said cassetteis under the control of said promoter in said plant cell.
 122. The DNAvector of claim 121, wherein the promoter is selected from the groupconsisting of nopaline synthase promoter (NOS), octopine synthasepromoter (OCS), cauliflower mosaic virus 19S promoter (CaMV19S),cauliflower mosaic virus 35S promoter (CaMV35S), ribulose1,5-bisphosphate carboxylase promoter (ssRUBISCO), and the figwortmosaic virus promoter (FMV).
 123. The DNA vector of claim 121, whereinthe promoter is selected from the group of root tissue enhanced or roottissue specific promoters consisting of the CaMV derived AS4 promoter,tobacco RB7 promoter, and the rice RC2 promoter.
 124. The DNA vector ofclaim 121, wherein said enzyme is approximately M_(r) 50,000.
 125. TheDNA vector of claim 121, wherein: said enzyme is isolated from anextract of a fungal species fermentation; and said extract exhibitscoleopteran insecticidal activity.
 126. The DNA vector of claim 125,wherein said insecticidal activity is independent of tedanalactamsynthase activity.
 127. The DNA vector of claim 125, wherein the fungalspecies is a Trichoderma.
 128. The DNA vector of claim 121, wherein said5′ non-translated leader sequence is obtained from a 70 kDa petunia heatshock protein (hsp70) gene.
 129. A DNA vector comprising: a promoterwhich functions in a plant cell to cause the production of an RNAsequence, operably linked to a polynucleotide cassette comprising; a 5′non-translated leader sequence; a structural gene encoding a lysineoxidase enzyme or proenzyme; and a DNA sequence which functions inplants to cause the addition of a 3′ non-translated polyadenylatednucleotide sequence to the 3′ end of said RNA sequence; whereinexpression of said cassette is under the control of said promoter insaid plant cell.
 130. The DNA vector of claim 129, wherein the promoteris selected from the group consisting of nopaline synthase promoter(NOS), octopine synthase promoter (OCS), cauliflower mosaic virus 19Spromoter (CaMV19S), cauliflower mosaic virus 35S promoter (CaMV35S),ribulose 1,5-bisphosphate carboxylase promoter (ssRUBISCO), and thefigwort mosaic virus promoter (FMV).
 131. The DNA vector of claim 129,wherein said promoter is selected from the group of root enhanced orspecific promoters consisting of the CaMV derived AS4 promoter, tobaccoRB7 promoter, and the rice RC2 promoter.
 132. The DNA vector of claim129, wherein the lysine oxidase enzyme is isolated from an extract of afungal species fermentation, and wherein said extract exhibitscoleopteran insecticidal activity.
 133. The DNA vector of claim 132,wherein the fungal species is a Trichoderma.
 134. The DNA vector ofclaim 129, wherein said 5′ non-translated leader sequence is obtainedfrom a 70 kDa petunia heat shock protein (hsp70) gene.
 135. Agenetically transformed seed containing a double stranded DNA molecule,said DNA molecule comprising: a first structural gene encoding a lysineoxidase enzyme or proenzyme; and a second structural gene encoding anenzyme that converts Δ1-piperideine-2-carboxylate to tedanalactam. 136.The genetically transformed seed of claim 135, wherein said secondstructural gene encodes an enzyme that is approximately M_(r) 50,000.137. The genetically transformed seed of claim 135, wherein: said secondstructural gene encodes an enzyme that is isolated from an extract of afungal species fermentation; and said extract exhibits coleopteraninsecticidal activity.
 138. The genetically transformed seed of claim137, wherein the insecticidal activity is independent of tedanalactamsynthase activity.
 139. The genetically transformed seed of claim 137,wherein the fungal species is a Trichoderma.
 140. A seed capable ofgermination into a plant, said plant containing a double stranded DNAmolecule, said DNA molecule comprising: a first structural gene encodinga lysine oxidase enzyme or proenzyme; and a second structural geneencoding an enzyme that converts Δ1-piperideine-2-carboxylate totedanalactam.
 141. The seed of claim 140, wherein said second structuralgene encodes an enzyme that is approximately M_(r) 50,000.
 142. The seedof claim 140, wherein: said second structural gene encodes an enzymethat is isolated from an extract of a fungal species fermentation; andsaid extract exhibits coleopteran insecticidal activity.
 143. The seedof claim 142 wherein the insecticidal activity is independent oftedanalactam synthase activity.
 144. The seed of claim 142, wherein thefungal species is a Trichoderma.
 145. A plant germinated from the seedof claim
 140. 146. A DNA vector comprising: a promoter which functionsin a plant cell to cause the production of an RNA sequence, operablylinked to a polynucleotide cassette comprising; an intron sequence; astructural gene encoding an enzyme that convertsΔ1-piperideine-2-carboxylate to tedanalactam; and a DNA sequence whichfunctions in plants to cause the addition of a 3′ non-translatedpolyadenylated nucleotide sequence to the 3′ end of said RNA sequence;wherein expression of said cassette is under the control of saidpromoter in said plant cell.
 147. The DNA vector of claim 146, whereinsaid promoter is selected from the group consisting of nopaline synthasepromoter (NOS), octopine synthase promoter (OCS), cauliflower mosaicvirus 19S promoter (CaMV19S), cauliflower mosaic virus 35S promoter(CaMV35S), ribulose 1,5-bisphosphate carboxylase promoter (ssRUBISCO),and the figwort mosaic virus promoter (FMV).
 148. The DNA vector ofclaim 146, wherein said promoter is selected from the group of rootenhanced or specific promoters consisting of the CaMV derived AS4promoter, tobacco RB7 promoter, and the rice RC2 promoter.
 149. The DNAvector of claim 146, wherein said enzyme is approximately M_(r) 50,000.150. The DNA vector of claim 146, wherein: said enzyme is isolated froman extract of a fungal species fermentation; and said extract exhibitscoleopteran insecticidal activity.
 151. The DNA vector of claim 150,wherein said insecticidal activity is independent of tedanalactamsynthase activity.
 152. The DNA vector of claim 150, wherein the fungalspecies is a Trichoderma.
 153. The DNA vector of claim 146, wherein saidintron is selected from the group consisting of the maize hsp70 intronand the rice actin intron.
 154. A DNA vector comprising: a) a promoterwhich functions in a plant cell to cause the production of an RNAsequence, operably linked to a polynucleotide cassette comprising; b) anintron sequence; c) a structural gene encoding a lysine oxidase enzymeor proenzyme; and d) a DNA sequence which functions in plants to causethe addition of a 3′ non-translated polyadenylated nucleotide sequenceto the 3′ end of said RNA sequence; wherein expression of said cassetteis under the control of said promoter in said plant cell.
 155. The DNAvector of claim 154, wherein said promoter is selected from the groupconsisting of nopaline synthase promoter (NOS), octopine synthasepromoter (OCS), cauliflower mosaic virus 19S promoter (CaMV19S),cauliflower mosaic virus 35S promoter (CaMV35S), ribulose1,5-bisphosphate carboxylase promoter (ssRUBISCO), and the figwortmosaic virus promoter (FMV).
 156. The DNA vector of claim 154, whereinsaid promoter is selected from the group of root enhanced or specificpromoters consisting of the CaMV derived AS4 promoter, tobacco RB7promoter, and the rice RC2 promoter.
 157. The DNA vector of claim 154,wherein: said lysine oxidase enzyme is isolated from an extract of afungal species fermentation; and said extract exhibits insecticidalactivity.
 158. The DNA vector of claim 157, wherein said fungal speciesis a Trichoderma.
 159. The DNA vector of claim 154, wherein said intronis selected from the group of specific introns consisting of the maizehsp70 intron and the rice actin intron.
 160. A DNA vector comprising: a)a first promoter which functions in a plant cell to cause the productionof a first RNA sequence, operably linked to a first polynucleotidecassette comprising; b) a first intron sequence; c) a first DNA sequenceencoding an amino acid sequence which functions in plants as a targetingsignal or transit peptide; d) a first structural gene encoding an aminoacid oxidase; and e) a first DNA sequence which functions in plants tocause the addition of a 3′ non-translated polyadenylated nucleotidesequence to the 3′ end of said RNA sequence; wherein expression of saidfirst cassette is under the control of said first promoter in said plantcell; and f) a second promoter which functions in a plant cell to causethe production of a second RNA sequence, operably linked to a secondpolynucleotide cassette comprising; g) a second intron sequence; h) asecond structural gene encoding an enzyme that convertsΔ1-piperideine-2-carboxylate to tedanalactam; and i) a second DNAsequence which functions in plants to cause the addition of a 3′non-translated polyadenylated nucleotide sequence to the 3′ end of saidRNA sequence; wherein expression of said second cassette is under thecontrol of said second promoter in said plant cell.
 161. The DNA vectorof claim 160, wherein said amino acid oxidase is a lysine oxidase. 162.The DNA vector of claim 160, wherein: said enzyme is isolated from anextract of a fungal species fermentation; and said extract exhibitscoleopteran insecticidal activity.
 163. The DNA vector of claim 162,wherein said insecticidal activity is independent of tedanalactamsynthase activity.
 164. The DNA vector of claim 163, wherein said fungalspecies is a Trichoderma.
 165. The DNA vector of claim 160, wherein saidfirst promoter and said second promoter are selected from the groupconsisting of nopaline synthase promoter (NOS), octopine synthasepromoter (OCS), cauliflower mosaic virus 19S promoter (CaMV19S),cauliflower mosaic virus 35S promoter (CaMV35S), ribulose1,5-bisphosphate carboxylase promoter (ssRUBISCO), and the figwortmosaic virus promoter (FMV).
 166. The DNA vector of claim 160, whereinsaid first promoter and said second promoter are selected from the groupof root enhanced or specific promoters consisting of the CaMV derivedAS4 promoter, tobacco RB7 promoter, and the rice RC2 promoter.
 167. TheDNA vector of claim 160, wherein said first intron and said secondintron are selected from the group of specific introns consisting of themaize hsp70 intron and the rice actin intron.
 168. The DNA vector ofclaim 160, wherein said targeting signal or transit peptide are selectedfrom the group consisting of a rice malate dehydrogenase amino terminalperoxisomal targeting signal and a maize ATP synthase beta subunitmitochondrial transit peptide.
 169. A method for identifying a geneencoding an enzyme comprising the steps of: a) contacting apolynucleotide probe and a heterologous polynucleotide sample to producea complex; b) detecting said complex; c) isolating said complex; and d)purifying the bound heterologous polynucleotide from said complex;wherein: a mixture of said enzyme and an amino acid oxidase providesinsecticidal activity; and said polynucleotide probe is selected fromthe group consisting of SEQ ID NO: 40, SEQ ID NO: 42, the complement ofSEQ ID NO: 40, and the complement of SEQ ID NO:
 42. 170. A method fordetecting a peptide comprising the steps of: a) contacting an antibodyand a sample to produce an antibody-peptide complex; and b) detectingsaid complex; wherein a mixture of said peptide and an amino acidoxidase provides insecticidal activity.
 171. The method of claim 170,wherein said sample comprises a composition derived from cells selectedfrom the group consisting of bacteria, plants, and fungi.
 172. Themethod of claim 171, wherein said fungi are members of the fungalspecies Trichoderma.
 173. A kit for detecting the presence of a peptidein a sample, said kit comprising a nucleic acid molecule packaged in acontainer; wherein: a mixture of said peptide and an amino acid oxidaseprovides insecticidal activity; and the nucleic acid molecule isselected from the group consisting of SEQ ID NO: 40, a derivative of SEQID NO: 40, or a fragment of SEQ ID NO:
 40. 174. The kit of claim 173,wherein said sample comprises a composition derived from cells selectedfrom the group consisting of bacteria, plants, and fungi.
 175. The kitof claim 174, wherein said fungi are members of the fungal speciesTrichoderma.
 176. A kit for detecting a peptide in a sample, said kitcomprising an antibody packaged in a container; wherein: said antibodybinds to SEQ ID NO: 40; said antibody binds to said peptide; and amixture of said peptide and an amino acid oxidase provides insecticidalactivity.
 177. The kit of claim 176, wherein said sample comprises acomposition derived from cells selected from the group consisting ofbacteria, plants, and fungi.
 178. The kit of claim 177, wherein saidfungi are members of the fungal species Trichoderma.